WO2024216096A1 - Cd46 binding vectors and uses thereof to treat cns disorders - Google Patents

Abstract

Described herein are hCD46-binding adenoviral vectors and the use of such vectors in the treatment of CNS disorders

Description

CD46 BINDING VECTORS AND USES THEREOF TO TREAT CNS DISORDERS

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under grants DC014093-07, NS110742-01, and TR004161-01 awarded by the National Institutes of Health. The government has certain rights in the invention.

PRIORITY

This application claims the benefit of priority of US provisional patent application No. 63/459,405, filed on April 14, 2023, the benefit of priority of which is claimed hereby, and which is incorporated by reference herein in its entirety.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

This application contains a sequence listing which has been submitted electronically in ST26 format and is hereby incorporated by reference in its entirety. Said ST26 file, created on April 10, 2024, is named “875242WOl.xml” and is 39,884 bytes in size.

BACKGROUND

Genetic variations in genes are major risk factors for a range of central nervous system (CNS) disorders. Genetic variations can result in a complex landscape of loss of function, gain of function, or a mixed gain/loss of function of proteins, which can result in either broad or cell typespecific dysfunction or degeneration. Despite the understanding of causes of CNS disorders, therapeutics that correct the root cause of dysfunction across the entire landscape has not been developed.

Viral vector gene therapy has emerged as potential treatment strategy for CNS disorders since viral vectors can restore gene function. They can correct the underlying cause of disease through stable expression of a therapeutic transgene via a single administration. Among the vectors utilized for gene therapy, recombinant Adeno-associated virus (AAV), lentiviral vectors (LVV) and recombinant Adenoviral vectors (Ad) have emerged as the vectors of choice. However, AAV and LVV have a packaging capacity of ~5 kb and ~9 kb respectively, rendering them incompatible to treat many CNS disorders requiring delivery of large or multiple gene constructs. Large transgene cassettes required to target many CNS disorders simply do not fit within an AAV and their size relative to LVV payload result in low viral titers, severely limiting utility. By contrast, Helper-dependent Adenovirus vectors (HdAd) that are adenoviral vectors devoid of all viral genes, have a large ~36 kb packaging capacity and can be easily produced to extremely high titers. HdAd is non-toxic and enables long-term correction of genetic disorders after a single dose in animal models. Currently, the most common Adenoviral vectors are of serotype 5 (Ad5) and infect cells via Coxsackie-Adenovirus Receptor (CAR). However, due to low CAR expression in neurons, these vectors are unable to transduce many neuronal cell types that are dysfunctional or degenerate in many CNS disorders, thus are not viable to treat many CNS disorders.

SUMMARY

Viral vector gene therapy has a role in treating central nervous system (CNS) disorders. Although adeno-associated virus vectors (AAV) have had success, their small packaging capacity limit their utility to treat the root cause of many CNS disorders. Adenoviral vectors (Ad) have tremendous potential for CNS gene therapy approaches. Currently, the most common vectors utilize the Group C Ad5 serotype capsid proteins, which rely on the Coxsackievirus-Adenovirus receptor (CAR) to infect cells. However, these Ad5 vectors are unable to transduce many neuronal cell types that are dysfunctional in many CNS disorders. The human CD46 (hCD46) receptor is widely expressed throughout the human CNS and is the primary attachment receptor for many Ad serotypes. Therefore, to overcome the current limitations of Ad vectors to treat CNS disorders, chimeric 1st generation Ad vectors were created that utilize the hCD46 receptor. Using a “humanized” hCD46 mouse model, it was demonstrated that these Ad vectors transduce cerebellar cell-types, including Purkinje cells, that are refractory to Ad5 transduction. Since Ad vector transduction properties are dependent on their capsid proteins, these chimeric 1st generation Ad vectors open new avenues for high-capacity helper-dependent adenovirus (HdAd) gene therapy approaches for cerebellar disorders and multiple neurological disorders.

Human CD46 (hCD46) interacting molecules on delivery vehicles, such as hCD46 binding adenoviral vectors, as disclosed herein, fill a need in basic research or gene therapy studies aimed to express large or multiple gene constructs including therapeutic constructs for CNS applications. The disclosure provides, in one embodiment, viral vector platforms with applicability to human gene therapy approaches for treatment of CNS disorders or CNS applications in basic research settings involving animal models and the use of the ligands that target the hCD46 receptor for non-viral gene therapy approaches or drug delivery.

To overcome the limitation of Ad5, Ad vector variants were developed that use an alternative and abundant human receptor. These vectors were characterized in the cerebellum of a humanized mouse model and variants identified with tropism for Purkinje Cells that are of high therapeutic value due to their involvement in various CNS disorders. The development of these vectors expands the utility of the adenoviral vector platform for treating cerebellar and other CNS disorders, for example, CACNA1A disorders, A-T, SCA-1, SCA-3, Friedreich ataxia and Huntington’s disease, particularly those that require expression of genes or transgene cassettes exceeding the capacities of AAV or LV. Thus, these vectors have translational applicability to humans for cerebellar and other CNS disorders.

This disclosure enables the preparation of, for example, Ad vectors and drug delivery systems, that can transduce certain CNS cell-types that underpin many CNS disorders. In addition, this disclosure enables the production of, for example, helper-dependent adenovirus (HdAd) and other Ad vectors that utilize hCD46 receptor for transduction of certain neuronal cell types to deliver up to about 36 kb of DNA cargo including large or multiple gene constructs in vivo. This would include native promoters. 5’ untranslated regions, 3’ untranslated region, cassettes that express RNAi to knock down mutant protein while restoring while type protein,/

In one embodiment, Ad5/21, Ad5/35, and A5/50 vectors were prepared that use hCD46 receptor for transduction. These vectors were used to demonstrate that they show altered tropism compared to the standard Ad5 vector. It was shown that hCD46 utilizing vectors were able to transduce a key neuronal cell type of the CNS that was not possible with Ad5. Ad5/21, Ad5/35 and A5/50 vectors transduced Purkinje cells of the cerebellum that become dysfunctional or degenerate in multiple degenerative disorders of the CNS. Thus, adenoviral vectors that utilize the CD46 receptor for viral vector gene therapy approaches to treat central nervous system disorders are provided.

The disclosure thus provides a delivery vehicle comprising a molecule that binds to hCD46, comprising: an adenovirus fiber/knob region that binds to hCD46 and optionally one or more prophylactic or therapeutic gene products or nucleic acid encoding one of more of the prophylactic or therapeutic gene products. In one embodiment, the delivery vehicle comprises a recombinant virus comprising the adenoviral fiber/knob region that binds to hCD46. In one embodiment, the delivery vehicle comprises a recombinant adenovirus comprising: a recombinant adenoviral genome that encodes the adenoviral tail and the fiber/knob region, e.g., which is El’ and/or E3‘. In one embodiment, the adenovirus genome is from a human adenovirus. In one embodiment, at least a portion of the adenovirus fiber/knob region is from a human adenovirus. In one embodiment, the adenovirus is helper dependent. In one embodiment, the fiber/knob region is from serotype 21. In one embodiment, the fiber/knob region is from serotype 35. In one embodiment, the fiber/knob region is from serotype 50. In one embodiment, the delivery vehicle further comprises a transgene encoding a prophylactic or therapeutic gene product. In one embodiment, the gene is a CACNA1 or ATM gene. In one embodiment, the delivery vehicle comprises a liposome. In one embodiment, the delivery vehicle of comprises a nanoparticle or microparticle. In one embodiment, the gene product comprises a protein. In one embodiment, the delivery vehicle comprises a liposome comprising the protein. In one embodiment, the delivery vehicle comprises a nanoparticle or microparticle comprising the protein. In one embodiment, the delivery vehicle encodes a plurality of prophylactic or therapeutic gene products. In one embodiment, the delivery vehicle is a rAAV or lentivirus vector.

Further provided is a pharmaceutical composition comprising the delivery vehicle and optionally a carrier.

In one embodiment, a method to prevent, inhibit or treat a disease or disorder of the central nervous system is provided, comprising: administering to a mammal in need thereof a composition comprising an effective amount of the of delivery vehicle or the pharmaceutical composition. In one embodiment, the composition is systemically administered. In one embodiment, the composition is locally administered. In one embodiment, the composition is administered to the central nervous system. In one embodiment, the composition is intracranially administered. In one embodiment, the composition is intrathecally administered. In one embodiment, the composition is intraci stemally administered. In one embodiment, the composition is intracerebrovascularly administered. In one embodiment, the mammal is a human, the mammal has an ataxia. In one embodiment, the mammal has Huntington’s disease. In one embodiment, the mammal has amyotrophic lateral sclerosis. In one embodiment, the mammal has a CaV2.1 channelopathy. In one embodiment, the mammal has familial hemiplegic migraine type 1 (FHM1), episodic ataxia type 2, or spinocerebellar ataxia type 6 (SCA6). In one embodiment, the gene product comprises CACNA1, Huntingtin, FMRI, ATM, ATXN2, or C9ORF72.

The vectors may be useful in monogenic CNS disorders, to express, for example, CACNA1A, which is useful in methods to prevent, inhibit or treat CaV 2.1 channelopathy, spinocerebellar ataxia type 6 (SCA6), familial hemiplegic migraine type 1 (FHM1), or episodic ataxia type 2 (EA2); ATM, which is useful in methods to prevent, inhibit or treat Ataxia Telangiectasia (AT); Huntingtin, which is useful in methods to prevent, inhibit or treat Huntington’s Disease; SCN2A, which is useful in methods to prevent, inhibit or treat Developmental and Epileptic Encephalopathy; N0TCH3, which is useful in methods to prevent, inhibit or treat CADASIL Syndrome; SPTBN2, which is useful in methods to prevent, inhibit or treat Spinocerebellar Ataxia type 5 (SCA5); ITPR1, which is useful in methods to prevent, inhibit or treat Spinocerebellar Ataxia type 16 (SCA16); SETX, which is useful in methods to prevent, inhibit or treat Ataxia with oculomotor apraxia 2 (AOA-2); VPS 13D, , which is useful in methods to prevent, inhibit or treat Spinocerebellar Ataxia, Autosomal Recessive 4 (SCAR4); ATXN1, which is useful in methods to prevent, inhibit or treat Spinocerebellar Ataxia type 1 (SCA1); ATXN3, which is useful in methods to prevent, inhibit or treat Spinocerebellar Ataxia type 3 (SCA3); ATXN7, which is useful in methods to prevent, inhibit or treat Noonan Syndrome Spinocerebellar Ataxia type 7 (SCA7); TTBK2, which is useful in methods to prevent, inhibit or treat Noonan Syndrome Spinocerebellar Ataxia type 11 (SCA11); PPP2R2B, which is useful in methods to prevent, inhibit or treat Noonan Syndrome Spinocerebellar Ataxia type 12 (SCA12); KCNC3, which is useful in methods to prevent, inhibit or treat Noonan Syndrome Spinocerebellar Ataxia type 13 (SCAB); KCND3, which is useful in methods to prevent, inhibit or treat Noonan Syndrome Spinocerebellar Ataxia type 19 (SCA19); ATPB3, which is useful in methods to prevent, inhibit or treat Noonan Syndrome Spinocerebellar Ataxia X-linked 1 (SCAX1); FXN, which is useful in methods to prevent, inhibit or treat Noonan Syndrome Friedrich Ataxia; MAPT, which is useful in methods to prevent, inhibit or treat Noonan Syndrome Frontotemporal Dementia; MECP2, which is useful in methods to prevent, inhibit or treat Noonan Syndrome Rett Syndrome; or PTPN11 and/or KRAS, which is useful in methods to prevent, inhibit or treat Noonan Syndrome.

The vectors are also useful to deliver multiple genes and/or replace chromosomal deletions in diseases such as Prader-Willi Syndrome, Angelman Syndrome, Schaaf-Yang Syndrome, DiGeorge Syndrome or Williams-Beuren Syndrome.

BRIEF DESCRIPTION OF THE FIGURES

FIG 1. Genomic map of El/E3-deleted Ad5, Ad5/21, Ad5/35, and Ad5/50 vectors. El and E3 -deleted Ad5 vector has been constructed to express the red fluorescent reporter protein mCherry under control of CMV promoter. Ad5/21, Ad5/35 and Ad5/50 vectors expressed the green fluorescent reporter protein mClover3. Ad5 shaft and knob were replaced with shaft and knob sequences of Ad21 or Ad35 or Ad50 viruses to generate Ad5/21 CMV mClover3, Ad5/35 CMV mClover3, and Ad5/50 CMV mclover3 vectors.

FIG. 2. Depiction of a novel Adenovirus vector that transduces Purkinje cells in a humanized mouse model. Since commonly used Adenovirus vectors do not transduce Purkinje cells, these vectors can be used in gene therapy approaches for cerebellar disorders and other neurological disorders, including those requiring expression of large transgenes.

FIG. 3. Fiber/knob sequences for Ad21, Ad35, and Ad50 (SEQ ID NOS: 13-15).

FIGS. 4A-4G. Mouse model and generation of viral vectors A/B.

Photomicrographs of RNA-ISH BaseScope Assay performed on the sagittal cerebellar sections of wild-type mouse (A) and hCD46tg mouse (B). N(wt/tg)=3/3. Black arrowheads indicate Purkinje cells (PC). Blue: hematoxylin, Red: chromogenic hCD46 probe signal, ML: molecular layer, PCL: Purkinje cell layer, GCL: granule cell layer. Dashed lines indicate PCL. C/D. Immunofluorescence staining performed on cerebellar sections from wild-type (C) and hCD46tg (D) mice labeling PC-specific molecular marker, Pcp2 (green) and hCD46 (red). Blue: DAPI. N(wt/tg)=l/l. E/F. Confirmation of chimeric vector generation by distinct restriction enzyme digestion patterns of PCR-amplified fiber/knob fragments derived from purified vectors. Agarose gel image demonstrating AflIII digestion patterns, indicating successful incorporation of Ad21, Ad35, or Ad50 shaft and knob sequences into Ad5 genome. Expected band sizes were observed for Ad5 (lane 1), Ad5/21 (lane 2), Ad5/35 (lane 3), and Ad5/50 (lane 4). G. Representative maps of viral genomes of E1ZE3 deleted 1st generation Ad5 and Group B chimeric vectors, depicting the modifications made to the shaft and knob regions of the fiber domain.

FIGS. 5A-5E. hCD46 dependent transduction of Purkinje cells by Group B chimeric vectors.

A. Illustration showcasing lobular administration of Ad5 individually co-mixed with Ad5/21, Ad5/35 or Ad5/50 into the mouse cerebellum and subsequent tissue processing steps. N(wt/tg)=3/3 for each chimeric vector. VP: viral particle. B. Fluorescent photomicrographs representing lack of Ad5 tropism to PCs in the cerebellar cortex of wild-type (left column) and hCD46tg (right column) mice. Red: Ad5 transduction signal (mCherry), Blue: Pcp2 immunofluorescence signal. ML: molecular layer, PCL: Purkinje cell layer, GCL: granule cell layer. Yellow arrowheads indicate non-transduced PCs, dashed lines indicate PCL. C/DZE. Fluorescent photomicrographs representing Ad5/21 (C), Ad5/35 (D), and Ad5/50 (E) tropism to PCs in the cerebellar cortex of wild-type (left column) and hCD46tg (right column) mice. Green: chimeric vector transduction signal (mClover3). White arrowheads indicate transduced PCs. Data is derived from co-injections of each Ad5/Group B vector with the Ad5 vector, where IxlO⁹ VP per vector was delivered in 1 pl volume into the cerebellar simple lobule.

FIGS. 6A-6D. Injection of chimeric vectors into the DCN transduces Purkinje cells via retrograde axonal transport.

A. Illustration of sagittal mouse cerebellum section depicting DCN injection site targeting PC axon terminals. N(tg)=3 for each chimeric vector. B. Fluorescent photomicrographs representing lack of Ad5 tropism to PCs in the cerebellar cortex of hCD46tg mice upon DCN injection. Red: Ad5 transduction signal (mCherry), Blue: Pcp2 immunofluorescence signal. ML: molecular layer, PCL: Purkinje cell layer, GCL: granule cell layer. Dashed lines indicate PCL. C/D. Fluorescent photomicrographs representing transduction of PCs by Ad5/35 (C) and Ad5/50 (D) in the cerebellar cortex of hCD46tg mice upon DCN injection. Green: chimeric vector transduction signal (mClover3). White arrowheads indicate transduced PCs. Data is derived from co-injections of each Ad5/Group B vector with the Ad5 vector, where IxlO⁹ VP per vector was delivered in 1 pl volume into the DCN. FIGS. 7A-7D. Ad5/35 and Ad5/50 transduce comparable number of Purkinje cells via

DCN.

A. Illustration of coronal mouse cerebellum section depicting DCN injection performed to compare Ad5/35 to Ad5/50 and margin sections obtained distant to the injection site (dashed lines). N(tg)=3. TU: transducing units. B. Quantification from sagittal serial sections of number of PCs transduced by either Ad5/35 or Ad5/50 under CMV promoter, ns: p=0.9678, Student’s t- test. Data are plotted as mean ± SEM. C. Stacked bar graph indicating the percentage of PCs transduced by each vector and the percentage co-transduced by both vectors. D. Tiled photomicrographs representing PC transduction at the lateral margin (left) and medial margin (middle) of the cerebellum. Magnified reproduction (right) of the white frame on the medial margin section. Green: Ad5/35 vector transduction signal (mClover3), Red: Ad5/50 vector transduction signal (mScarlet), Blue: DAPI, ML: molecular layer, GCL: granule cell layer. Data is derived from co-injections of the Ad5/35 vector with the Ad5/50 vector, where 2xl0⁷ TU per vector was delivered in 1 pl volume in to the DCN.

FIGS. 8A-8D. PC-specific L7-6 promoter increases transduction efficiency.

A. Illustration of coronal mouse cerebellum section depicting DCN injection performed to compare CMV promoter to L7-6 promoter under the context of Ad5/50. N(tg)=3. Dashed lines indicate section obtained from proximity to the injection site. TU: transducing units. B. Quantification of number of PCs from sagittal serial sections transduced by Ad5/50 under either CMV or L7-6 promoters. *: p=0.0173, Student’s t-test. Data are plotted as mean ± SEM. C. Stacked bar graph indicating the percentage of PCs transduced by each vector and the percentage co-transduced by both vectors. D. Tiled photomicrograph representing Ad5/50 tropism at the injection site section (left) and magnified images corresponding to the lobule frame (middle) and DCN frame (right). Green: Ad5/50 CMV vector transduction signal (mClover3), Red: Ad5/50 L7- 6 vector transduction signal (mScarlet), Blue: DAPI, M 794 L: molecular layer, GCL: granule cell layer, DCN: deep cerebellar nuclei. Data is derived from co-injections of the Ad5/50 CMV vector with the Ad5/50 L7-6 vector, where 2xl0⁷ TU per vector was delivered in 1 pl volume into the DCN.

FIGS. 9A-9C. Ad5, Ad5/35, and Ad5/50 transduce Bergmann glia in a hCD46 independent manner.

A. Fluorescent photomicrographs representing Ad5 tropism to Bergmann glia in wild type (left column) and hCD46tg (right column) mice. Red: Ad5 transduction signal (mCherry), Blue: slOOp immunofluorescence signal. ML: molecular layer, PCL: Purkinje cell layer, GCL: granule cell layer. B/C. Fluorescent photomicrographs representing Ad5/35 (B) and Ad5/50 (C) tropism to Bergmann glia in wild-type (left column) and hCD46tg (right column) mice. White arrowheads indicate transduced Bergmann glia. Green: chimeric vector transduction signal (mClover3). N(wt/tg) = 3/3 for each chimeric vector. Data is derived from co-inj ections of each Ad5/Group B vector with the Ad5 vector, where 1x109 VP per vector was delivered in 1 pl volume into the cerebellar simple lobule.

FIGS. 10A-10C. Ad5/35 and Ad5/50 transduce granule cells in a hCD46 independent manner.

A. Fluorescent photomicrographs of granule cell layer representing lack of Ad5 tropism to granule cells in wild-type (left column) and hCD46tg (right column) mice. Red: Ad5 transduction signal (mCherry), Blue: NeuN immunofluorescence signal. GCL: granule cell layer B/C. Fluorescent photomicrographs of granule cell layer representing Ad5/35 (B) and Ad5/50 (C) tropism to granule cells in wild-type (left column) and hCD46tg (right column) mice. White arrowheads indicate transduced granule cells. Green: chimeric vector transduction signal (mClover3). N(wt/tg) = 3/3 for each chimeric vector. Data is derived from co-inj ections 817 of each Ad5/Group B vector with the Ad5 vector, where IxlO⁹ VP per vector was delivered in 1 pl volume into the cerebellar simple lobule.

FIGS. 11A-11D. Ad5/35 and Ad5/50 transduce mossy fibers in a hCD46 independent manner.

A. Tiled photomicrograph indicating retrograde transduction of mossy fibers leading to labeling of cell somata in various nuclei in pons and medulla. ECU: external cuneate nucleus, RN: reticular nuclei, PRN: pontine reticular nucleus. Dashed line traces the pontomedullary junction (left). Magnified image corresponding to the solid frame (middle) shows the transduction patterns at the injected simple lobule. Dashed frame indicates transduced mossy fiber terminals by Ad5/50 (green-mClover3) but not by Ad5 (red-mCherry) in a cerebellar lobule distant to the injected lobule (right). ML: molecular layer, PCL: Purkinje cell layer, GCL: granule cell layer. Blue: DAPI. Dashed lines in magnified reproductions indicate PCL. Representative image originates from a hCD46tg mouse. B. Fluorescent photomicrographs of granule cell layer representing lack of Ad5 transduction of mossy fiber terminals in wild-type (left) and hCD46tg (right) mice. Red: Ad5 transduction signal (mCherry), Blue: vGlutl immunofluorescence signal. C/D. Fluorescent photomicrographs of granule cell layer representing Ad5/35 (C) and Ad5/50 (D) transduction of mossy fiber terminals in wild-type (left) and hCD46tg (right) mice. White arrowheads indicate transduced mossy fiber terminals. Green: chimeric vector transduction signal (mClover3). N(wt/tg) = 3/3 for each chimeric vector. Data is derived from co-inj ections of each Ad5/Group B vector with the Ad5 vector, where 1x109 VP per vector was delivered in 1 pl volume into the cerebellar simple lobule.

DETAILED DESCRIPTION

Genetic variations in genes are major risk factors for a range of central nervous system (CNS) disorders. The genetic variations can result in a complex landscape of loss of function, gain of function, or a mixed gain/loss of function of proteins, which can result in either broad or cell type-specific dysfunction or degeneration. Despite the understanding of causes of CNS disorders, therapeutics that correct the root cause of dysfunction across the entire landscape has not been developed.

Since the causes of CNS disorders are due to genetic variation, viral vector gene therapy has emerged a potential treatment strategy. Viral vectors have the potential to restore gene function to treat many CNS disorders. They can correct the underlying cause of disease through stable expression of a therapeutic transgene via a single administration. Among the vectors utilized for gene therapy, recombinant Adeno-associated virus (AAV) (Li & Samullski, 2020), lentiviral vectors (LVV) (Poletti & Mavilio, 2021) and recombinant Adenoviral vectors (Ad) (Watanabe et al., 2021) have emerged as the vectors of choice. These vectors are in various stages of clinical trials to assess their efficacy in treating a wide variety of disorders in humans, with AAV having the most clinical success (Dunbar et al., 2018; Pupo et al., 2022). However, AAV and LVV have a packaging capacity of about 5 kb and about 9 kb respectively, rendering them incompatible to treat many CNS disorders requiring delivery of large or multiple gene constructs. Large transgene cassettes required to target many CNS disorders simply do not fit within an AAV and their size relative to LVV payload result in low viral titers, severely limiting utility (Poletti & Mavilio, 2021). By contrast, Helper-dependent Adenovirus vectors (HdAd) may be ideal. HdAd is devoid of all viral genes, giving these vectors an improved safety profile over earlier generation Ad vectors that are already being used in human clinical trials (Watanabe et al., 2021). HdAd is nontoxic and enables long-term correction of genetic disorders after a single dose in animal models (Brunetti-Pierri & Ng, 2017; Vetrini & Ng, 2010). Importantly, HdAd has a large about 36 kb packaging capacity and can be easily produced to extremely high titers (Palmer & Ng, 2011; Montesinos et al., 2016). Therefore, HdAd overcomes the major limitations of other viral vector gene therapy approaches and shows immense promise to treat the root cause of CNS disorders.

Currently, the vast majority of HdAd vectors are based on the Ad5 serotype, which relies on the Coxsackievirus-Adenovirus receptor (CAR) to infect cells. However, these vectors are unable to transduce many neuronal cell types that are dysfunctional or degenerate in many CNS disorders (Kaemmerer et al., 2000; Sato et al., 2004). Thus, these vectors are not viable to treat many CNS disorders. Therefore, development of a HdAd variant that transduces key CNS cell types is of critical importance. The Human CD46 (hCD46) receptor is widely expressed throughout the human CNS and the human CD46 receptor is the primary attachment receptor for many Ad serotypes (Arnberg, 2012). Therefore, to overcome the current limitation of HdAd and earlier generations of Ad vectors in treating CNS disorders, Ad vectors were developed that contain the Fiber/Knob domain from adenovirus serotypes that use hCD46 as their primary attachment receptor (Gaggar et al., 2003; Wang et al., 2008). It was demonstrated that these novel vectors transduce CNS cell types that are refractory to Ad5 infection. The availability of Ad vectors that target the hCD46 receptor greatly expands the utility for HdAd and Ad viral vectors in general for gene therapy approaches in the human CNS and for basic research applications in animal models.

Definitions

The term “about” is used herein to mean a value that is ±10% of the recited value.

As used herein, by “administering” is meant a method of giving a dosage of a composition described herein to a subject. The compositions utilized in the methods described herein can be administered by any suitable route, including, for example, by inhalation, nebulization, aerosolization, intranasally, intratracheally, intrabronchially, orally, parenterally (e.g., intravenously, subcutaneously, or intramuscularly), orally, nasally, rectally, topically, orbuccally. In some embodiments, a composition described herein is administered in aerosolized particles intratracheally and/or intrabronchially using an atomizer sprayer (e.g., with a MADgic® laryngotracheal mucosal atomization device). The compositions utilized in the methods described herein can also be administered locally or systemically. The method of administration can vary depending on various factors (e.g., the components of the composition being administered and the severity of the condition being treated).

A “control element” or “control sequence” is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3' direction) from the promoter. Promoters include Adenovirus promoters, AAV promoters, e.g., P5, P19, P40 and AAV ITR promoters, as well as heterologous promoters. An “expression vector” is a vector comprising a region which encodes a polypeptide of interest or other gene product, and is used for effecting the expression of the protein or product in an intended target cell. An expression vector also comprises control elements operatively linked to the encoding region to facilitate expression of the protein in the target. The combination of control elements and a gene or genes to which they are operably linked for expression is sometimes referred to as an “expression cassette,” a large number of which are known and available in the art or can be readily constructed from components that are available in the art.

A “gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated.

The term “gene delivery” refers to the introduction of an exogenous polynucleotide into a cell for gene transfer, and may encompass targeting, binding, uptake, transport, localization, replicon integration and expression.

The term “gene transfer” refers to the introduction of an exogenous polynucleotide into a cell which may encompass targeting, binding, uptake, transport, localization and replicon integration, but is distinct from and does not imply subsequent expression of the gene.

The term “gene expression” or “expression” refers to the process of gene transcription, translation, and post-translational modification.

“Heterologous” means derived from a genotypically distinct entity from that of the rest of the entity to which it is compared. For example, a polynucleotide introduced by genetic engineering techniques into a different cell type is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide).

“Host cells,” “cell lines,” “cell cultures,” “packaging cell line” and other such terms denote eukaryotic cells, e.g., mammalian cells, such as human cells, useful in the present disclosure. These cells can be used as recipients for recombinant vectors, viruses or other transfer polynucleotides, and include the progeny of the original cell that was transduced. It is understood that the progeny of a single cell may not necessarily be completely identical (in morphology or in genomic complement) to the original parent cell.

An “isolated” plasmid, virus, or other substance refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially prepared from. Thus, for example, an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichments of the embodiments of this disclosure are increasingly more some. Thus, for example, a 2-fold enrichment is some, 10-fold enrichment is more some, 100-fold enrichment is more some, 1000-fold enrichment is even more some.

As used herein, the term “operable linkage” or “operably linked” refers to a physical or functional juxtaposition of the components so described as to permit them to function in their intended manner. More specifically, for example, two DNA sequences operably linked means that the two DNAs are arranged (cis or trans) in such a relationship that at least one of the DNA sequences is able to exert a physiological effect upon the other sequence. For example, an enhancer and/or a promoter can be operably linked with a transgene (e.g., a therapeutic transgene).

The term “polynucleotide” refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. A polynucleotide may comprise modified nucleotides, such as methylated or capped nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the disclosure described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

The terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably and refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single-or double-stranded form. For the purposes of the present disclosure, these terms are not to be construed as limiting with respect to the length of a polymer. The terms can encompass known analogues of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties (e.g., phosphorothioate backbones). In general, an analogue of a particular nucleotide has the same base-pairing specificity; i.e., an analogue of A will base-pair with T.

The terms “polypeptide” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, acetylation, phosphorylation, lipidation, or conjugation with a labeling component. Polypeptides such as “ABCA4” and the like, when discussed in the context of gene therapy and compositions therefor, refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof that retains the desired biochemical function of the intact protein. Similarly, references to genes for use in gene therapy (typically referred to as “transgenes” to be delivered to a recipient cell), include polynucleotides encoding the intact polypeptide or any fragment or genetically engineered derivative possessing the desired biochemical function.

By “pharmaceutical composition” is meant any composition that contains a therapeutically or biologically active agent (e.g., a polynucleotide comprising a transgene or a portion thereof), either incorporated into a viral vector (e.g., an rAAV vector) or independent of a viral vector (e.g., incorporated into a liposome, microparticle, or nanoparticle)) that is suitable for administration to a subject. Any of these formulations can be prepared by well-known and accepted methods of art. See, for example, Remington: The Science and Practice of Pharmacy (21st ed.), ed. A.R. Gennaro, Lippincott Williams & Wilkins, 2005, and Encyclopedia of Pharmaceutical Technology, ed. J. Swarbrick, Informa Healthcare, 2006, each of which is hereby incorporated by reference.

By “pharmaceutically acceptable diluent, excipient, carrier, or adjuvant” is meant a diluent, excipient, carrier, or adjuvant which is physiologically acceptable to the subject while retaining the therapeutic properties of the pharmaceutical composition with which it is administered.

“Recombinant,” as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction and/or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature. A recombinant virus is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.

By “reference” is meant any sample, standard, or level that is used for comparison purposes. A “normal reference sample” or a “wild-type reference sample” can be, for example, a sample from a subject not having the disorder (e.g., retinal dysfunction). A “positive reference” sample, standard, or value is a sample, standard, value, or number derived from a subject that is known to have a disorder, which may be matched to a sample of a subject by at least one of the following criteria: age, weight, disease stage, and overall health.

The terms “subject” and “patient” are used interchangeably herein to refer to any mammal (e.g., a human, a primate, a cat, a dog, a ferret, a cow, a horse, a pig, a goat, a rat, or a mouse). For example, the subject is a human.

A “terminator” refers to a polynucleotide sequence that tends to diminish or prevent read- through transcription (i.e., it diminishes or prevent transcription originating on one side of the terminator from continuing through to the other side of the terminator). The degree to which transcription is disrupted is typically a function of the base sequence and/or the length of the terminator sequence. In particular, as is well known in numerous molecular biological systems, particular DNA sequences, generally referred to as “transcriptional termination sequences” are specific sequences that tend to disrupt read-through transcription by RNA polymerase, presumably by causing the RNA polymerase molecule to stop and/or disengage from the DNA being transcribed. Typical example of such sequence-specific terminators include polyadenylation (“polyA”) sequences, e.g., SV40 polyA. In addition to or in place of such sequence-specific terminators, insertions of relatively long DNA sequences between a promoter and a coding region also tend to disrupt transcription of the coding region, generally in proportion to the length of the intervening sequence. This effect presumably arises because there is always some tendency for an RNA polymerase molecule to become disengaged from the DNA being transcribed, and increasing the length of the sequence to be traversed before reaching the coding region would generally increase the likelihood that disengagement would occur before transcription of the coding region was completed or possibly even initiated. Terminators may thus prevent transcription from only one direction (“uni-directional” terminators) or from both directions (“bi-directional” terminators), and may be comprised of sequence-specific termination sequences or sequence-non-specific terminators or both. A variety of such terminator sequences are known in the art; and illustrative uses of such sequences within the context of the present disclosure are provided below.

A “therapeutic gene,” “prophylactic gene,” “target polynucleotide,” “transgene,” “gene of interest” and the like generally refer to a gene or genes to be transferred using a vector. Typically, in the context of the present disclosure, such genes are located within the delivery vector. Target polynucleotides can be used in this disclosure to generate vectors for a number of different applications. Such polynucleotides include, but are not limited to: (i) polynucleotides encoding proteins useful in other forms of gene therapy to relieve deficiencies caused by missing, defective or sub-optimal levels of a structural protein or enzyme; (ii) polynucleotides that are transcribed into anti-sense molecules; (iii) polynucleotides that are transcribed into decoys that bind transcription or translation factors; (iv) polynucleotides that encode cellular modulators such as cytokines; (v) polynucleotides that can make recipient cells susceptible to specific drugs, such as the herpes virus thymidine kinase gene; (vi) polynucleotides for cancer therapy, such as E1A tumor suppressor genes or p53 tumor suppressor genes for the treatment of various cancers; and (vii) polynucleotides for gene editing (e.g., CRISPR). To effect expression of the transgene in a recipient host cell, it is in one embodiment operably linked to a promoter, either its own or a heterologous promoter. A large number of suitable promoters are known in the art, the choice of which depends on the desired level of expression of the target polynucleotide; whether one desires constitutive expression, inducible expression, cell-specific or tissue-specific expression, etc. The vector may also contain a selectable marker. By “therapeutically effective amount” is meant the amount of a composition administered to improve, inhibit, or ameliorate a condition of a subject, or a symptom of a disorder or disease, in a clinically relevant manner. Any improvement in the subject is considered sufficient to achieve treatment. In one embodiment, an amount sufficient to treat is an amount that reduces, inhibits, or prevents the occurrence or one or more symptoms of a disease or disorder or is an amount that reduces the severity of, or the length of time during which a subject suffers from, one or more symptoms of the disease or disorder (e.g., by at least about 10%, about 20%, or about 30%, or by at least about 50%, about 60%, or about 70%, or by at least about 80%, about 90%, about 95%, about 99%, or more, relative to a control subject that is not treated with a composition described herein). An effective amount of the pharmaceutical composition used to practice the methods described herein varies depending upon the manner of administration and the age, body weight, and general health of the subject being treated. A physician or researcher can decide the appropriate amount and dosage regimen.

In particular, a "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount is also one in which any toxic or detrimental effects of the vector(s)are outweighed by the therapeutically beneficial effects.

As used herein, an "effective amount" or a "therapeutically effective amount" of a set of vectors, e.g., a recombinant virus encoding a portion of a gene product, refers to an amount of the set that alleviates, in whole or in part, symptoms associated with the disorder or condition, or halts or slows further progression or worsening of those symptoms, or prevents or provides prophylaxis for the disorder or condition, e.g., an amount that is effective to prevent, inhibit or treat in the individual one or more symptoms.

“Transduction” or “transducing” as used herein, are terms referring to a process for the introduction of an exogenous polynucleotide, e.g., a transgene in Ad, into a host cell leading to expression of the polynucleotide, e.g., the transgene in the cell. Altered expression or persistence of a polynucleotide introduced via the vector can be determined by methods well known to the art including, but not limited to, protein expression, e.g., by ELISA, flow cytometry and Western blot, measurement of DNA and RNA production by hybridization assays, e.g., Northern blots, Southern blots and gel shift mobility assays, or quantitative or non-quantitative reverse transcription, polymerase chain reaction (PCR), or digital droplet PCR assays.

“Treatment” of an individual or a cell is any type of intervention in an attempt to alter the natural course of the individual or cell at the time the treatment is initiated, e.g., eliciting a prophylactic, curative or other beneficial effect in the individual. For example, treatment of an individual may be undertaken to decrease or limit the pathology caused by any pathological condition, including (but not limited to) an inherited or induced genetic deficiency, infection by a viral, bacterial, or parasitic organism, a neoplastic or aplastic condition, or an immune system dysfunction such as autoimmunity or immunosuppression. Treatment includes (but is not limited to) administration of a composition, such as a pharmaceutical composition, and administration of compatible cells that have been treated with a composition. Treatment may be performed either prophylactically or therapeutically; that is, either prior or subsequent to the initiation of a pathologic event or contact with an etiologic agent. Treatment may reduce one or more symptoms of a pathological condition. Detecting an improvement in, or the absence of, one or more symptoms of a disorder, indicates successful treatment.

For example, "treating" or "treatment" within the meaning herein refers to an alleviation of symptoms associated with a disorder or disease, "inhibiting" means inhibition of further progression or worsening of the symptoms associated with the disorder or disease, and "preventing" refers to prevention of the symptoms associated with the disorder or disease.

A “variant” refers to a polynucleotide or a polypeptide that is substantially homologous to a native or reference polynucleotide or polypeptide. For example, a variant polynucleotide may be substantially homologous to a native or reference polynucleotide, but which has a polynucleotide sequence different from that of the native or reference polynucleotide because of one or a plurality of deletions, insertions, and/or substitutions. In another example, a variant polypeptide may be substantially homologous to a native or reference polypeptide, but which has an amino acid sequence different from that of the native or reference polypeptide because of one or a plurality of deletions, insertions, and/or substitutions. Variant polypeptide-encoding polynucleotide sequences encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference polynucleotide sequence, but that encode a variant protein or fragment thereof that retains activity. A wide variety of mutagenesis approaches are known in the art and can be applied by a person of ordinary skill in the art.

A variant polynucleotide or polypeptide sequence may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, identical to a native or reference sequence. The degree of homology (percent identity) between a native and a variant sequence can be determined, for example, by comparing the two sequences using freely available computer programs commonly employed for this purpose on the world wide web (e.g., BLASTp or BLASTn with default settings). A “vector” as used herein refers to a macromolecule or association of macromolecules that comprises or associates with a polynucleotide and which can be used to mediate delivery of the polynucleotide to a cell, either in vitro or in vivo. Illustrative vectors include, for example, plasmids, viral vectors, liposomes and other gene delivery vehicles. The polynucleotide to be delivered, sometimes referred to as a transgene, may comprise a coding sequence of interest in gene therapy (such as a gene encoding a protein of therapeutic or interest), a coding sequence of interest in vaccine development (such as a polynucleotide expressing a protein, polypeptide or peptide suitable for eliciting an immune response in a mammal), and/or a selectable or detectable marker.

As used herein, "individual" (as in the subject of the treatment) means a mammal. Mammals include, for example, humans; non-human primates, e.g., apes and monkeys; and nonprimates, e.g., dogs, cats, rats, mice, cattle, horses, sheep, and goats. Non-mammals include, for example, fish and birds.

The term "disease" or "disorder" are used interchangeably, and are used to refer to diseases or conditions, e.g., wherein lack of or reduced amounts of a specific gene product, or a mutant gene product, plays a role in the disease such that a therapeutically beneficial effect can be achieved by supplementing, e.g., to at least 1%, 10%, 25%, 50% or more of normal levels.

"Substantially" as the term is used herein means completely or almost completely; for example, a composition that is "substantially free" of a component either has none of the component or contains such a trace amount that any relevant functional property of the composition is unaffected by the presence of the trace amount, or a compound is "substantially pure" is there are only negligible traces of impurities present.

The term "sequence" refers to a nucleotide sequence of any length, which can be DNA or RNA; can be linear, circular or branched and can be either single-stranded or double stranded.

A "homologous, non-identical sequence" refers to a first sequence which shares a degree of sequence identity with a second sequence, but whose sequence is not identical to that of the second sequence. For example, a polynucleotide comprising the wild-type sequence of a mutant gene is homologous and non-identical to the sequence of the mutant gene. In certain embodiments, the degree of homology between the two sequences is sufficient to allow homologous recombination therebetween, utilizing normal cellular mechanisms.

A "disease associated gene" is one that is defective in some manner in, for example, a monogenic disease.

An "exogenous" molecule is a molecule that is not normally present in a cell but can be introduced into a cell by one or more genetic, biochemical or other methods. "Normal presence in the cell" is determined with respect to the particular developmental stage and environmental conditions of the cell. Thus, for example, a molecule that is present only during embryonic development of a specific tissue or cell is an exogenous molecule with respect to an adult tissue or cell. An exogenous molecule can comprise, for example, a functioning version of a malfunctioning endogenous molecule or a malfunctioning version of a normally functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, such as is generated by a combinatorial chemistry process, or a macromolecule such as a protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules. Nucleic acids include DNA and RNA, can be single-or double-stranded; can be linear, branched or circular; and can be of any length. Nucleic acids include those capable of forming duplexes, as well as triplexforming nucleic acids.

An exogenous molecule can be the same type of molecule as an endogenous molecule, e.g., an exogenous protein or nucleic acid. For example, an exogenous nucleic acid can comprise an infecting viral genome, a plasmid or episome introduced into a cell, or a chromosome that is not normally present in the cell. Methods for the introduction of exogenous molecules into cells are known to those of skill in the art and include, but are not limited to, lipid-mediated transfer (e.g., liposomes, including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer and viral vector-mediated transfer. An exogenous molecule can also be the same type of molecule as an endogenous molecule but derived from a different species than the cell is derived from. For example, a human nucleic acid sequence may be introduced into a cell line originally derived from a mouse or hamster.

By contrast, an "endogenous" molecule is one that is normally present in a particular cell at a particular developmental stage under particular environmental conditions. For example, an endogenous nucleic acid can comprise a chromosome, the genome of a mitochondrion, chloroplast or other organelle, or a naturally occurring episomal nucleic acid.

“Gene expression” or “expression” refers to the process of gene transcription, translation, and post-translational modification.

An “infectious” virus or viral particle is one that comprises a polynucleotide component which it is capable of delivering into a cell for which the viral species is trophic. The term does not necessarily imply any replication capacity of the virus. A “transcriptional regulatory sequence” refers to a genomic region that controls the transcription of a gene or coding sequence to which it is operably linked. Transcriptional regulatory sequences of use generally include at least one transcriptional promoter and may also include one or more enhancers and/or terminators of transcription.

A “terminator” refers to a polynucleotide sequence that tends to diminish or prevent read- through transcription (i.e., it diminishes or prevent transcription originating on one side of the terminator from continuing through to the other side of the terminator). The degree to which transcription is disrupted is typically a function of the base sequence and/or the length of the terminator sequence. In particular, as is well known in numerous molecular biological systems, particular DNA sequences, generally referred to as “transcriptional termination sequences” are specific sequences that tend to disrupt read-through transcription by RNA polymerase, presumably by causing the RNA polymerase molecule to stop and/or disengage from the DNA being transcribed. Typical example of such sequence-specific terminators include polyadenylation (“polyA”) sequences, e.g., SV40 polyA. In addition to or in place of such sequence-specific terminators, insertions of relatively long DNA sequences between a promoter and a coding region also tend to disrupt transcription of the coding region, generally in proportion to the length of the intervening sequence. This effect presumably arises because there is always some tendency for an RNA polymerase molecule to become disengaged from the DNA being transcribed, and increasing the length of the sequence to be traversed before reaching the coding region would generally increase the likelihood that disengagement would occur before transcription of the coding region was completed or possibly even initiated. Terminators may thus prevent transcription from only one direction (“uni-directional” terminators) or from both directions (“bi-directional” terminators), and may be comprised of sequence-specific termination sequences or sequence-non-specific terminators or both. A variety of such terminator sequences are known in the art; and illustrative uses of such sequences within the context of the present disclosure are provided below.

"Transformed" or "transgenic" is used herein to include any host cell or cell line, which has been altered or augmented by the presence of at least one recombinant DNA sequence. The host cells are typically produced by transfection with a DNA sequence in a plasmid expression vector, as an isolated linear DNA sequence, or infection with a recombinant viral vector.

The term “sequence homology” means the proportion of base matches between two nucleic acid sequences or the proportion amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, e.g., 50%, the percentage denotes the proportion of matches over the length of a selected sequence that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, 6 bases or less or 2 bases or less. When using oligonucleotides as probes or treatments, the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally not less than 17 target base matches out of 20 possible oligonucleotide base pair matches (85%); not less than 9 matches out of 10 possible base pair matches (90%), or not less than 19 matches out of 20 possible base pair matches (95%).

Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less or 2 or less. Alternatively, two protein sequences (or polypeptide sequences derived from them of at least 30 amino acids in length) are homologous, as this term is used herein, if they have an alignment score of at more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater. The two sequences or parts thereof are more homologous if their amino acids are greater than or equal to 50% identical when optimally aligned using the ALIGN program.

The term “corresponds to” is used herein to mean that a polynucleotide sequence is structurally related to all or a portion of a reference polynucleotide sequence, or that a polypeptide sequence is structurally related to all or a portion a reference polypeptide sequence, e.g., they have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, sequence identity. In contradistinction, the term “complementary to” is used herein to mean that the complementary sequence is homologous to all or a portion of a reference polynucleotide sequence. For illustration, the nucleotide sequence “TATAC” corresponds to a reference sequence “TATAC” and is complementary to a reference sequence “GTATA”.

The term “sequence identity” means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term “percentage of sequence identity” means that two polynucleotide sequences are identical (i.e., on a nucleotide- by-nucleotide basis) over the window of comparison. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. The terms “substantial identity” as used herein denote a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence that has at least 85 percent sequence identity, at least 90 to 95 percent sequence identity, at least 99 percent sequence identity as compared to a reference sequence over a comparison window of at least 20 nucleotide positions, frequently over a window of at least 20-50 nucleotides, wherein the percentage of sequence identity is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.

“Conservative” amino acid substitutions are, for example, aspartic-glutamic as polar acidic amino acids; lysine/arginine/histidine as polar basic amino acids; leucine/isoleucine/methionine/valine/alanine/glycine/proline as non-polar or hydrophobic amino acids; serine/ threonine as polar or uncharged hydrophilic amino acids. Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting polypeptide. Whether an amino acid change results in a functional polypeptide can readily be determined by assaying the specific activity of the polypeptide. Naturally occurring residues are divided into groups based on common side-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gin, his, lys, arg; (5) residues that influence chain orientation: gly, pro; and (6) aromatic; trp, tyr, phe.

The disclosure also envisions polypeptides with non-conservative substitutions. Nonconservative substitutions entail exchanging a member of one of the classes described above for another.

“Nucleic acid sequence” is intended to encompass a polymer of DNA or RNA, i.e., a polynucleotide, which can be single-stranded or double-stranded and which can contain nonnatural or altered nucleotides. The terms “nucleic acid” and “polynucleotide” as used herein refer to a polymeric form of nucleotides of any length, either ribonucleotides (RNA) or deoxyribonucleotides (DNA). These terms refer to the primary structure of the molecule, and thus include double- and single-stranded DNA, and double- and single-stranded RNA. The terms include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and modified polynucleotides such as, though not limited to, methylated and/or capped polynucleotides.

Gene Therapy Vectors

The disclosure provides a gene therapy vector comprising a nucleic acid sequence which encodes one or more gene products, e.g., one or more proteins. The disclosure further provides a method of using the vector to introduce genes into certain cells. Various aspects of the gene therapy vector and method are discussed below. Although each parameter is discussed separately, the gene therapy vector and methods comprise combinations of the parameters set forth below. Accordingly, any combination of parameters can be used according to the gene therapy vector and the method.

A “gene therapy vector” is thus any molecule or composition that has the ability to carry a heterologous nucleic acid sequence into a suitable host cell where synthesis of the encoded protein takes place. Typically, a gene therapy vector is a nucleic acid molecule that has been engineered, using recombinant DNA techniques that are known in the art, to incorporate the heterologous nucleic acid sequence. Desirably, the gene therapy vector is comprised of DNA. Examples of suitable DNA-based gene therapy vectors include plasmids and viral vectors. However, gene therapy vectors that are not based on nucleic acids, such as liposomes, are also known and used in the art. The gene therapy vector can be based on a single type of nucleic acid (e.g., a plasmid) or non-nucleic acid molecule (e.g., a lipid or a polymer). The gene therapy vector can be integrated into the host cell genome or can be present in the host cell in the form of an epi some.

Gene delivery vectors within the scope of the disclosure include, but are not limited to, isolated nucleic acid, e.g., plasmid-based vectors which may be extrachromosomally maintained, and viral vectors, e.g., recombinant adenovirus, retrovirus, lentivirus, herpesvirus, poxvirus, papilloma virus, or adeno-associated virus, including viral and non-viral vectors, e.g., nucleic acid based vectors, which are present in liposomes, e.g., neutral or cationic liposomes, such as DOSPA/DOPE, DOGS/DOPE or DMRIE/DOPE liposomes, and/or associated with other molecules such as DNA-anti-DNA antibody-cationic lipid (DOTMA/DOPE) complexes. Exemplary viral gene delivery vectors are described below. Gene delivery vectors may be administered via any route including, but not limited to, intracranial, intrathecal, intramuscular, buccal, rectal, intravenous or intracoronary administration, and transfer to cells may be enhanced using electroporation and/or iontophoresis, and/or scaffolding such as extracellular matrix or hydrogels, e.g., a hydrogel patch. In one embodiment, the gene therapy vector is a viral vector. Suitable viral vectors include, for example, retroviral vectors, lentivirus vectors, herpes simplex virus (HSV)-based vectors, parvovirus-based vectors, e.g., adeno-associated virus (AAV)-based vectors, AAV- adenoviral chimeric vectors, and adenovirus-based vectors. These viral vectors can be prepared using standard recombinant DNA techniques described in, for example, Sambrook et al., Molecular Cloning, a Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994).

In one embodiment, viral vectors include, but are not limited to, reoviruses, adenoviruses, adeno-associated viruses, papovaviruses, parvoviruses, picornaviruses, and enteroviruses of any suitable origin (e.g., of animal origin (e.g., avian or mammalian) and desirably of human origin). Other suitable viral vectors are known in the art and are well characterized. Examples of such viral vectors are described in, for example, Fields et al., VIROLOGY Lippincott-Raven (3rd ed. (1996) and 4th ed. (2000)); ENCYCLOPEDIA OF VIROLOGY, R. G. Webster et al., eds., Academic Press (2nd ed., 1999); FUNDAMENTAL VIROLOGY, Fields et al., eds., Lippincott-Raven (3rd ed., 1995); Levine, “Viruses,” Scientific American Library No. 37 (1992);

MEDICAL VIROLOGY, D. O. White et al., eds., Academic Press (2nd ed. 1994); and INTRODUCTION TO MODERN VIROLOGY, Dimock, N. J. et al., eds., Blackwell Scientific Publications, Ltd. (1994). A viral vector may be derived from, or based on, a virus that normally infects animals, such as mammals (such as humans). Adenoviral (Ad) vectors based on human or non-human primate adenoviruses may be used as viral vectors.

In one embodiment, the gene therapy vector is a non-integrating viral vector, e.g., Ad, AAV, integration-deficient lentiviral vectors (IDLVs), and others.

Retroviral vectors

Retroviral vectors exhibit several distinctive features including their ability to stably and precisely integrate into the host genome providing long-term transgene expression. These vectors can be manipulated ex vivo to eliminate infectious gene particles to minimize the risk of systemic infection and patient-to-patient transmission. Pseudotyped retroviral vectors can alter host cell tropism.

Lentiviruses

Lentiviruses are derived from a family of retroviruses that include human immunodeficiency virus and feline immunodeficiency virus. However, unlike retroviruses that only infect dividing cells, lentiviruses can infect both dividing and nondividing cells. Although lentiviruses have specific tropisms, pseudotyping the viral envelope with vesicular stomatitis virus yields virus with a broader range (Schnepp et al., Meth, Mol, Med., 69:427 (2002)).

In one embodiment, lentiviral vectors may be employed. These vectors do not encode any viral product, as the viral proteins are provided in trans from several packaging plasmids to split the original viral genome. Accessory genes, often responsible for pathogenic features, have been progressively removed from the production system. Vectors have also been made self-inactivating (SIN) by deleting the transcriptional promoter/enhancer from the 3’ LTR in the transfer plasmid; this deletion is copied onto the 5’ end of the vector during the reverse transcription cycle, abolishing expression from the viral LTR. SIN vectors are therefore dependent on an internal promoter to provide transgenic expression Additionally, high-efficiency lentiviral transduction can be achieved with IDLVs, produced through the use of integrase mutations that specifically prevent proviral integration, resulting in the generation of increased levels of circular vector episomes. Lacking replication signals, lentiviral episomes mediate transient transduction in dividing cells and stable expression in quiescent cells. It is also possible to use retroviral vectors for so-called retrovirus particle-mediated mRNA transfer (RMT), whereby vector mutants unable to start reverse transcription are instead transiently translated.

Adenoviral vectors

Adenoviral vectors may be rendered replication-incompetent by deleting the early (El A and E1B) genes responsible for viral gene expression from the genome and are stably maintained into the host cells in an extrachromosomal form. These vectors [El -] have the ability to transfect both replicating and nonreplicating cells and, in particular, these vectors have been shown to efficiently infect cardiac myocytes in vivo, e.g., after direction injection or perfusion. El- adenoviral vectors have been shown to result in transient expression of therapeutic genes in vivo, peaking at 7 days and lasting approximately 4 weeks. These vectors have a packaging capacity of ~8 kb.

Helper-dependent Adenoviral vectors (HdAd) are constructed by removing all viral sequences from the vector genome. Only the packaging sequence and inverted terminal repeats are retained. This allows for long lasting therapeutic gene expression (months to years) upon single administration and an improved capacity of up to ~38 kb.

Adenovirus vector features include efficient delivery to dividing and non-dividing cells, retention as non-integrated nuclear linear episomes, and high but transient expression. In addition, adenoviral vectors can be produced at very high titers, allowing efficient gene therapy with small volumes of virus. Adenoviruses are a family of DNA viruses with an icosahedral, 70-100nm in diameter, non-enveloped capsid engulfing a double-stranded (ds) DNA genome. These viruses can infect quiescent and dividing cells and replicate in the cell nucleus. Human Ad serotypes from a range of >50 Ad subdivisions/clades, with a typical Ad5 vector genome of ~36kb encoding genes that are expressed before (Early, E) and after (Late, L) viral replication. Early transcription units encode proteins required for viral transactivation and host-virus interactions. Non-human primate (NHP) adenoviruses from chimpanzees, bonobos and gorillas and various other species may be employed in the methods. Exemplary non-human including simian, e.g., gorilla, chimpanzee, and rhesus adenoviruses include but are not limited to GC44, GC45, GC46, Pan5, Pan6, Pan7, Pan9, GRAd, AdC7, AdC21, AdC6, S AdV-11, SAdV-16, PanAd3, ChAd23, ChAd24, sAdl6, sAdl9, ChAdOxl, AdC68 sAd33, RhAd51, RhAd52 or RhAd53, as well as adenoviruses disclosed in Abbink et al. (J. Virol,, 89: 1512 (2015)), the disclosure of which is incorporated by reference herein. Conventional Ad vectors are constructed by substituting the deleted regions of the adenovirus genome with the transgene cassette of interest.

Adeno-associated virus vectors

Recombinant adeno-associated viruses (rAAV) are derived from nonpathogenic parvoviruses and produce transgene expression lasting months to years in most systems. Moreover, like adenovirus, adeno-associated virus vectors also have the capability to infect replicating and nonreplicating cells and are believed to be nonpathogenic to humans.

Plasmid DNA vectors

Plasmid DNA is often referred to as "naked DNA" to indicate the absence of a more elaborate packaging system. Direct injection of plasmid DNA to myocardial cells in vivo has been accomplished. Plasmid-based vectors are relatively nonimmunogenic and nonpathogenic, with the potential to stably integrate in the cellular genome, resulting in long-term gene expression in postmitotic cells in vivo. For example, expression of secreted angiogenesis factors after muscle injection of plasmid DNA, despite relatively low levels of focal transgene expression, has demonstrated significant biologic effects in animal models and appears promising clinically (Isner, Nature, 415:234 (2002)). Furthermore, plasmid DNA is rapidly degraded in the blood stream; therefore, the chance of transgene expression in distant organ systems is negligible. Plasmid DNA may be delivered to cells as part of a macromolecular complex, e.g., a liposome or DNA-protein complex, and delivery may be enhanced using techniques including electroporation. Poxyirus

Poxviral vectors, including vaccinia Poxviral vectors features include large- capacity dsDNA viruses (>25 kb of foreign DNA) and transient expression of proteins. Poxviruses are members of the family Poxviridae. They are dsDNA viruses about 200-400n min length with a genome of about 190kb, which is flanked by ~10kb ITRs, and exist in two forms: an intracellular naked virion (INV) and an extracellular enveloped virion (EEV). Transcription and DNA replication occur in the cytoplasm, where the progeny DNA is generated by the synthesis and resolution of large concatemeric molecules. Recombinant poxviruses have the transgene of interest commonly inserted by homologous recombination and driven by a poxviral promoter rather than a constitutive viral or mammalian promoter, since they are cytoplasmatic viruses and encode their own RNA polymerase. Modified Vaccinia virus Ankara (MV A) is licensed as third- generation vaccine against smallpox. Recombinant MVAs (rMVAs) can be used for protein production and as vaccines against infectious diseases, cancer and other pathologies.

Other non-integrating viral vector systems include herpes virus vectors, and particularly those based on HSV-1. Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV).

A number of non-viral methods for nucleic acid delivery have been developed, which can be classified as physical or chemical. Physical methods include the use of ultrasound or electrical currents to temporarily increase the permeability of target cells (sonoporation and electroporation, respectively), direct injection of DNA into single cells, ballistic propulsion of DNA-coated particles and hydrodynamic gene delivery involving the rapid injection of a large volume of DNA solution (8-10% of body weight). Gene delivery by physical methods is fairly simple but offers no protection from nucleases for the nucleic acid.

In contrast, chemical carriers typically encapsulate nucleic acids thereby protecting the payload from nucleases. Chemical gene delivery vectors usually employ a cationic species to condense the anionic nucleic acids and in the process form nanoparticles for delivery. Cationic liposomes have been extensively studied and are among the most widely used non-viral vectors. Later, addition of cationic polymers (producing so-called lipopolyplex) was shown to enhance gene delivery. Mechanistically, the liposome likely provides the mechanism for endosomal escape whilst the polymer enables efficient condensation and packaging of the nucleic acid therefore forming small, stable, discrete, and homogenous nanoparticles. Further attempts at improving non-viral formulations have been made with the addition of components to improve bioavailability in vivo through shielding of complexes using polyethylene glycols, to enhance cell-specific targeting using targeting moieties, to aid endosomal escape using fusogenic lipids or pH sensitive polymers, and to improve nuclear entry using nuclear targeting sequences or nuclear localization signal-containing peptides.

Electroporation technologies like nucleofection mediate efficient delivery of DNA and mRNA.

Suitable expression constructs include those designed for propagation and expansion or for expression or both. Examples of suitable expression constructs include plasmids, phagemids, cosmids, viruses, and other vehicles derived from viral or bacterial sources. Any of these expression constructs can be manipulated to include a nucleic acid sequence and can be prepared using standard recombinant DNA techniques described in, e.g., Sambrook et al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994).

Plasmids are genetically engineered circular double-stranded DNA molecules and can be designed to contain an expression cassette comprising a nucleic acid molecule encoding, for example, ATM. By complexing the plasmid with liposomes, the efficiency of gene transfer in general is improved. While the liposomes used for plasmid-mediated gene transfer strategies have various compositions, they are typically synthetic cationic lipids. Advantages of plasmidliposome complexes include their ability to transfer large nucleic acid sequences and their relatively low immunogenicity. While plasmids are suitable for use in the disclosure, the expression construct may be a viral vector.

Exemplary Adenovirus Vectors

Adenovirus is a 36 kb double-stranded DNA virus that efficiently transfers DNA in vivo to a variety of different target cell types. The Ad vector can be produced in high titers and can efficiently transfer DNA to replicating and non-replicating cells. The Ad vector genome can be generated using any species, strain, subtype, mixture of species, strains, or subtypes, or chimeric adenovirus as the source of vector DNA. Adenoviral stocks that can be employed as a source of adenovirus can be amplified from the adenoviral serotypes 1 through 51, which are currently available from the American Type Culture Collection (ATCC, Manassas, Va.), or from any other serotype of adenovirus available from any other source. For instance, an adenovirus can be of subgroup A (e.g., serotypes 12, 18, and 31), subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, 35, and 50), subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g., serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and 42-47), subgroup E (serotype 4), subgroup F (serotypes 40 and 41), or any other adenoviral serotype. Given that the human adenovirus serotype 5 (Ad5) genome has been completely sequenced, the adenoviral vector is described herein with respect to the Ad5 serotype. The Ad vector can be any adenoviral vector capable of growth in a cell, which is in some significant part (although not necessarily substantially) derived from or based upon the genome of an adenovirus. The Ad vector can be based on the genome of any suitable wild-type adenovirus. The Ad vector may be derived from the genome of a wild-type adenovirus of group C, especially of serotype 2 or 5. Ad vectors are well known in the art and are described in, for example, U.S. Pat. Nos. 5,559,099, 5,712,136, 5,731,190, 5,837,511, 5,846,782, 5,851,806, 5,962,311, 5,965,541, 5,981,225, 5,994,106, 6,020,191, and 6,113,913, International Patent Applications WO 95/34671, WO 97/21826, and WO 00/00628, and Thomas Shenk, “Adenoviridae and their Replication,” and M. S. Horwitz, “Adenoviruses,” Chapters 67 and 68, respectively, in Virology, B. N. Fields et al., eds., 3d ed., Raven Press, Ltd., New York (1996).

The Ad vector may be replication deficient. By “replication-deficient” is meant that the Ad vector comprises a genome that lacks at least one replication-essential gene function. A deficiency in a gene, gene function, or gene or genomic region, as used herein, is defined as a deletion of sufficient genetic material of the viral genome to impair or obliterate the function of the gene whose nucleic acid sequence was deleted in whole or in part. Replication-essential gene functions are those gene functions that are required for replication (i.e., propagation) of a replication-deficient Ad vector. Replication-essential gene functions are encoded by, for example, the adenoviral early regions (e.g., the El, E2, and E4 regions), late regions (e.g., the L1-L5 regions), genes involved in viral packaging (e.g., the IVa2 gene), and virus-associated RNAs (e.g., VA-RNA I and/or VA-RNA II). The replication-deficient Ad vector may comprise an adenoviral genome deficient in two or more gene functions required for viral replication. The two or more regions of the adenoviral genome may be selected from the group consisting of the El, E2, and E4 regions. The replication-deficient adenoviral vector may comprise a deficiency in at least one replication-essential gene function of the El region (denoted an El -deficient adenoviral vector). The El region of the adenoviral genome comprises the El A region and the E1B region. The El A and E1B regions comprise nucleic acid sequences coding for multiple peptides by virtue of RNA splicing. A deficiency of a gene function encoded by either or both of the El A and/or E1B regions of the adenoviral genome (e.g., a peptide that performs a function required for replication) is considered a deficiency of a gene function of the El region in the context of the disclosure. In addition to such a deficiency in the El region, the recombinant adenovirus also can have a mutation in the major late promoter (MLP), as discussed in International Patent Application WO 00/00628. The vector may be deficient in at least one replication-essential gene function of the El region and at least part of the nonessential E3 region (e.g., an Xba I deletion of the E3 region) (denoted an El/E3-deficient adenoviral vector). The adenoviral vector may be “multiply deficient,” meaning that the adenoviral vector is deficient in one or more gene functions required for viral replication in each of two or more regions of the adenoviral genome. For example, the aforementioned El-deficient or El/E3-deficient Ad vector can be further deficient in at least one replication-essential gene function of the E4 region (denoted an El/E4-deficient adenoviral vector). An adenoviral vector deleted of the entire E4 region can elicit a lower host immune response.

Alternatively, the Ad vector lacks replication-essential gene functions in all or part of the El region and all or part of the E2 region (denoted an El/E2-deficient adenoviral vector). Ad vectors lacking replication-essential gene functions in all or part of the El region, all or part of the E2 region, and all or part of the E3 region also are contemplated herein. If the Ad vector is deficient in a replication-essential gene function of the E2A region, the vector in one embodiment does not comprise a complete deletion of the E2A region, which is less than about 230 base pairs in length. Generally, the E2A region of the adenovirus codes for a DBP (DNA binding protein), a polypeptide required for DNA replication. DBP is composed of 473 to 529 amino acids depending on the viral serotype. It is believed that DBP is an asymmetric protein that exists as a prolate ellipsoid consisting of a globular Ct with an extended Nt domain. Studies indicate that the Ct domain is responsible for DBP's ability to bind to nucleic acids, bind to zinc, and function in DNA synthesis at the level of DNA chain elongation. However, the Nt domain is believed to function in late gene expression at both transcriptional and post-transcriptional levels, is responsible for efficient nuclear localization of the protein, and also may be involved in enhancement of its own expression. Deletions in the Nt domain between amino acids 2 to 38 have indicated that this region is important for DBP function (Brough et al., Virology, 196, 269-281 (1993)). While deletions in the E2A region coding for the Ct region of the DBP have no effect on viral replication, deletions in the E2A region which code for amino acids 2 to 38 of the Nt domain of the DBP impair viral replication. The multiply replication-deficient adenoviral vector may contain this portion of the E2A region of the adenoviral genome. In particular, for example, the desired portion of the E2A region to be retained is that portion of the E2A region of the adenoviral genome which is defined by the 5' end of the E2A region, specifically positions Ad5(23816) to Ad5(24032) of the E2A region of the adenoviral genome of serotype Ad5.

The Ad vector can be deficient in replication-essential gene functions of only the early regions of the adenoviral genome, only the late regions of the adenoviral genome, and both the early and late regions of the adenoviral genome. The adenoviral vector also can have essentially the entire adenoviral genome removed (denoted a helper-dependent adenoviral vector [HdAd]), in which case it may be preferred that at least either the viral (i.e., adenoviral) inverted terminal repeats (Ad ITRs) and one or more promoters or the Ad ITRs and a packaging signal are left intact (i.e., an adenoviral amplicon). The larger the region of the adenoviral genome that is removed, the larger the piece of exogenous nucleic acid sequence that can be inserted into the genome. For example, given that the adenoviral genome is 36 kb, by leaving the Ad ITRs and one or more promoters intact, the exogenous insert capacity of the adenovirus is approximately 35 kb. Alternatively, a multiply deficient Ad vector that contains only an Ad ITR and a packaging signal effectively allows insertion of an exogenous nucleic acid sequence of approximately 37-38 kb. Of course, the inclusion of a spacer element in any or all of the deficient adenoviral regions will decrease the capacity of the adenoviral vector for large inserts. Suitable replication-deficient Ad vectors, including multiply deficient Ad vectors, are disclosed in U.S. Pat. Nos. 5,851,806 and 5,994,106 and International Patent Applications WO 95/34671 and WO 97/21826. An adenoviral vector for use in the methods is that described in International Patent Application WO 02/00906.

It should be appreciated that the deletion of different regions of the Ad vector can alter the immune response of a mammal exposed to the Ad vector. In particular, the deletion of different regions can reduce the inflammatory response generated by the Ad vector. Furthermore, the Ad vector's coat protein can be modified so as to decrease the Ad vector's ability or inability to be recognized by a neutralizing antibody directed against the wild-type coat protein, as described in International Patent Application WO 98/40509.

The adenoviral vector, when multiply replication-deficient, especially in replicationessential gene functions of the El and E4 regions, e.g., includes a spacer element to provide viral growth in a complementing cell line similar to that achieved by singly replication deficient Ad vectors, particularly an Ad vector comprising a deficiency in the E4 region. A spacer sequence is defined in the disclosure as any sequence of sufficient length to restore the size of the adenoviral genome to approximately the size of a wild-type adenoviral genome, such that the Ad vector is efficiently packaged into viral particles. The spacer element can contain any sequence or sequences which are of the desired length. The spacer element sequence can be coding or noncoding and native or non-native with respect to the adenoviral genome, but does not restore the replication-essential function to the deficient region. The spacer can be of any suitable size, desirably at least about 15 base pairs (e.g., between about 15 base pairs and about 12,000 base pairs), about 100 base pairs to about 10,000 base pairs, about 500 base pairs to about 8,000 base pairs, about 1,500 base pairs to about 6,000 base pairs, or about 2,000 to about 3,000 base pairs. The size of the spacer is limited only by the size of the insert that the Ad vector will accommodate (e.g., approximately 38 kb). In the absence of a spacer, production of fiber protein and/or viral growth of the multiply replication-deficient Ad vector is reduced by comparison to that of a singly replication-deficient Ad vector. However, inclusion of the spacer in at least one of the deficient adenoviral regions, e.g., the E4 region, can counteract this decrease in fiber protein production and viral growth. The use of a spacer in an Ad vector is described in U.S. Pat. No. 5,851,806.

The Ad vector may contain a packaging domain. The packaging domain can be located at any position in the adenoviral genome, so long as the adenoviral genome is packaged into adenoviral particles. The packaging domain may be located downstream of the El region. The packaging domain may be located downstream of the E4 region. The replication-deficient Ad vector may lack all or part of the El region and the E4 region. A spacer may be inserted into the E4 region, a desired exogenous nucleic acid sequence of interest (e.g., a nucleic acid sequence encoding MECP2) is located in the El region, and the packaging domain is located downstream of the E4 region. By relocating the packaging domain, the amount of potential overlap between the Ad vector and the cellular/helper virus genome used to propagate the Ad vector is reduced so as to reduce the probability of obtaining a replication-competent Ad vector.

The coat proteins of the Ad vector can be manipulated to alter the binding specificity of the resulting adenoviral particle. Suitable modifications to the coat proteins include, but are not limited to, insertions, deletions, or replacements in the adenoviral fiber, penton, pIX, pllla, pVI, or hexon proteins, or any suitable combination thereof, including insertions of various native or non-native ligands into portions of such coat proteins. Examples of Ad vectors with modified binding specificity are described in, e.g., U.S. Pat. Nos. 5,871,727, 5,885,808, and 5,922,315. Modified Ad vector particles include those described in, for example, Wickham et al., J. Virol., 71(10), 7663-9 (1997), Cripe et al., Cancer Res., 61(7), 2953-60 (2001), van Deutekom et al., J. Gene Med., 1(6), 393-9 (1999), McDonald et al., J. Gene Med., 1(2), 103-10 (1999), Staba et al., Cancer Gene Ther., 7(1), 13-9 (2000), Wickham, Gene Ther., 'll!), 110-4 (2000), Kibbe et al., Arch. Surg., 135(2), 191-7 (2000), Harari et al., Gene Ther., 6(5), 801-7 (2000), Bouri et al., Hum Gene Ther., 10(10), 1633-40 (1999), Wickham et al., Nat. BiotechnoL, 14(11), 1570-3 (1996), Wickham et al., Cancer Immunol. Immunother., 45(3-4), 149-51 (1997), and Wickham et al., Gene Ther., 2(10), 750-6 (1995), and U.S. Pat. Nos. 5,559,099; 5,712,136; 5,731,190; 5,770,442; 5,801,030; 5,846,782; 5,962,311; 5,965,541; 6,057,155; 6,127,525; and 6,153,435; and International Patent Applications WO 96/07734, WO 96/26281, WO 97/20051, WO 98/07865, WO 98/07877, WO 98/40509, WO 98/54346, WO 00/15823, and WO 01/58940.

Replication-deficient Ad vectors are typically produced in complementing cell lines that provide gene functions not present in the replication-deficient Ad vectors, but required for viral propagation, at appropriate levels in order to generate high titers of viral vector stock. A cell line complements for at least one and optionally all replication-essential gene functions not present in a replication-deficient adenovirus. The complementing cell line can complement for a deficiency in at least one replication-essential gene function encoded by the early regions, late regions, viral packaging regions, virus-associated RNA regions, or combinations thereof, including all adenoviral functions (e.g., to enable propagation of adenoviral amplicons, which comprise minimal adenoviral sequences, such as only Ad ITRs and the packaging signal or only Ad ITRs and an adenoviral promoter). The complementing cell line complements for a deficiency in at least one replication-essential gene function (e.g., two or more replication-essential gene functions) of the El region of the adenoviral genome, particularly a deficiency in a replication-essential gene function of each of the E1A and E1B regions. In addition, the complementing cell line can complement for a deficiency in at least one replication-essential gene function of the E2 (particularly as concerns the adenoviral DNA polymerase and terminal protein) and/or E4 regions of the adenoviral genome. Desirably, a cell that complements for a deficiency in the E4 region comprises the E4-ORF6 gene sequence and produces the E4-ORF6 protein. Such a cell desirably comprises at least ORF6 and no other ORF of the E4 region of the adenoviral genome. The cell line may be further characterized in that it contains the complementing genes in a non-overlapping fashion with the adenoviral vector, which minimizes, and practically eliminates, the possibility of the vector genome recombining with the cellular DNA. Accordingly, the presence of replication- competent adenoviruses (RCA) is minimized if not avoided in the vector stock, which, therefore, is suitable for certain therapeutic purposes, especially gene therapy purposes. The lack of RCA in the vector stock avoids the replication of the Ad vector in non-complementing cells. The construction of complementing cell lines involves standard molecular biology and cell culture techniques, such as those described by Sambrook et al. (1989), supra, and Ausubel et al. (1984), supra. Complementing cell lines for producing adenoviral vectors include, but are not limited to, 293 cells (described in, e.g., Graham et al., J. Gen. Virol., 36, 59-72 (1977)), PER.C6 cells (described in, e.g., International Patent Application WO 97/00326, and U.S. Pat. Nos. 5,994,128 and 6,033,908), and 293-ORF6 cells (described in, e.g., International Patent Application WO 95/34671 and Brough et al., J. Virol., 71, 9206-9213 (1997)).

Expression Cassettes for Nucleic Acid Based Delivery

The selection of an expression construct for use in the disclosure may depend on a variety of factors such as, for example, the host, immunogenicity of the expression construct, the desired duration of protein production, the target cell, and the like. As each type of expression construct has distinct properties, a researcher has the freedom to tailor the disclosure to any particular situation. Moreover, more than one type of expression construct can be used, if desired. Accordingly, the nucleic acid molecule encoding, for example, CACNA1A is operably linked to regulatory sequences necessary for expression, especially a promoter. A “promoter” is a DNA sequence that directs the binding of RNA polymerase and thereby promotes RNA synthesis. A nucleic acid sequence is “operably linked” to a promoter when the promoter is capable of directing transcription of that nucleic acid sequence. A promoter can be native or non-native to the nucleic acid sequence to which it is operably linked.

Any promoter (i.e., whether isolated from nature or produced by recombinant DNA or synthetic techniques) can be used in connection with the disclosure to provide for transcription of a particular nucleic acid sequence. The promoter may be capable of directing transcription in a eukaryotic (desirably mammalian) cell. The functioning of the promoter can be altered by the presence of one or more enhancers and/or silencers present on the vector. “Enhancers” are cisacting elements of DNA that stimulate or inhibit transcription of adjacent genes. An enhancer that inhibits transcription also is termed a “silencer.” Enhancers differ from DNA-binding sites for sequence-specific DNA binding proteins found only in the promoter (which also are termed “promoter elements”) in that enhancers can function in either orientation, and over distances of up to several kilobase pairs (kb), even from a position downstream of a transcribed region.

The vector may employ a viral promoter. Suitable viral promoters are known in the art and include, for instance, cytomegalovirus (CMV) promoters, such as the CMV immediate-early promoter, promoters derived from human immunodeficiency virus (HIV), such as the HIV long terminal repeat promoter, Rous sarcoma virus (RSV) promoters, such as the RSV long terminal repeat, mouse mammary tumor virus (MMTV) promoters, HSV promoters, such as the Lap2 promoter or the herpes thymidine kinase promoter (Wagner et al., 7W45, 78, 144-145 (1981)), promoters derived from SV40 or Epstein Barr virus, an adeno-associated viral promoter, such as the p5 promoter, and the like. The viral promoter may be an adenoviral promoter, such as the Ad2 or Ad5 major late promoter and tripartite leader, a CMV promoter, or an RSV promoter.

Many of the above-described promoters are constitutive promoters. Instead of being a constitutive promoter, the promoter can be an inducible promoter, i.e., a promoter that is up- and/or down-regulated in response to appropriate signals. Examples of suitable inducible promoter systems include, but are not limited to, the IL-8 promoter, the metallothionine inducible promoter system, the bacterial lacZYA expression system, the tetracycline expression system, and the T7 polymerase system. Further, promoters that are selectively activated at different developmental stages (e.g., globin genes are differentially transcribed from globin-associated promoters in embryos and adults) can be employed. The promoter sequence that regulates expression of the nucleic acid sequence can contain at least one heterologous regulatory sequence responsive to regulation by an exogenous agent. The regulatory sequences may be responsive to exogenous agents such as, but not limited to, drugs, hormones, or other gene products. For example, the regulatory sequences, e.g., promoter, may be responsive to glucocorticoid receptor-hormone complexes, which, in turn, enhance the level of transcription of a therapeutic peptide or a therapeutic fragment thereof.

One of ordinary skill in the art will appreciate that each promoter drives transcription, and, therefore, protein expression, differently with respect to the time and amount of protein produced. For example, the CMV promoter is characterized as having peak activity shortly after transduction, i.e., about 24 hours after transduction, then quickly tapering off. On the other hand, the RSV promoter's activity increases gradually, reaching peak activity several days after transduction, and maintains a high level of activity for several weeks. Indeed, sustained expression driven by an RSV promoter has been observed in all cell types studied, including, for instance, liver cells, lung cells, spleen cells, diaphragm cells, skeletal muscle cells, and cardiac muscle cells. Thus, a promoter can be selected for use in the disclosure by matching its particular pattern of activity with the desired pattern and level of expression of a nucleic acid sequence of interest. Alternatively, a hybrid promoter can be constructed which combines the desirable aspects of multiple promoters. For example, a CMV-RSV hybrid promoter combining the CMV promoter's initial rush of activity with the RSV promoter's high maintenance level of activity may be employed. It is also possible to select a promoter with an expression profile that can be manipulated by an investigator.

With respect to promoters, nucleic acid sequences, selectable markers, and the like, located on an expression construct, such elements can be present as part of a cassette, either independently or coupled. In the context of the disclosure, a “cassette” is a particular base sequence that possesses functions, which facilitate subcloning, and recovery of nucleic acid sequences (e.g., one or more restriction sites) or expression (e.g., polyadenylation or splice sites) of particular nucleic acid sequences.

Construction of a nucleic acid sequence operably linked to regulatory sequences necessary for expression is well within the skill of the art (see, for example, Sambrook et al. (1989), supra). With respect to the expression of nucleic acid sequences according to the disclosure, the ordinary skilled artisan is aware that different genetic signals and processing events control levels of nucleic acids and proteins/peptides in a cell, such as, for instance, transcription, mRNA translation, and post-transcriptional processing. Transcription of DNA into RNA requires a functional promoter, as described herein. Protein expression is dependent on the level of RNA transcription that is regulated by DNA signals, and the levels of DNA template. Similarly, translation of mRNA requires, at the very least, an AUG initiation codon, which is usually located within 10 to 100 nucleotides of the 5' end of the message. Sequences flanking the AUG initiator codon have been shown to influence its recognition by eukaryotic ribosomes, with conformity to a perfect Kozak consensus sequence resulting in optimal translation (see, e.g., Kozak, J. Mol. BioL, 196, 947-950 (1987)). Also, successful expression of an exogenous nucleic acid in a cell can require post-translational modification of a resultant protein. Thus, production of a protein can be affected by the efficiency with which DNA (or RNA) is transcribed into mRNA, the efficiency with which mRNA is translated into protein, and the ability of the cell to carry out post-translational modification. These are all factors of which the ordinary skilled artisan is aware and is capable of manipulating using standard means to achieve the desired end result.

Along these lines, to optimize protein production, the nucleic acid molecule may further comprise a polyadenylation site following the coding region of the nucleic acid sequence. Also, the proper transcription signals (and translation signals, where appropriate) may be correctly arranged such that the nucleic acid sequence will be properly expressed in the cells into which it is introduced. Moreover, if the nucleic acid sequence encodes a protein or peptide, which is a processed or secreted protein or acts intracellularly, e.g., the nucleic acid sequence further comprises the appropriate sequences for processing, secretion, intracellular localization, and the like.

It will be appreciated that the expression construct can comprise multiple nucleic acid molecules. For example, the expression construct can comprise multiple copies of a nucleic acid molecule, each copy operably linked to a different promoter or to identical promoters. Moreover, any nucleic acid molecule described herein can be altered from its native form to increase or decrease a desired effect (e.g., to increase its therapeutic effect). For example, a cytoplasmic form of a nucleic acid molecule can be converted to a secreted form by incorporating a signal peptide into the encoded gene product.

Delivery Vehicles

Delivery vehicles include, for example, viral vectors, microparticles, nanoparticles, liposomes and other lipid-containing complexes, and other macromolecular complexes capable of mediating delivery of a gene or protein or other product to a host cell, e.g., to provide for recombinant expression of a polypeptide encoded by the gene. Vehicles can also comprise other components or functionalities that further modulate gene or protein delivery and/or gene expression, or that otherwise provide beneficial properties. Such other components include, for example, components that influence binding or targeting to cells (including components that mediate cell-type or tissue-specific binding); components that influence uptake of the vehicle by the cell; components that influence localization of the transferred gene or protein within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the gene. Such components also might include markers, such as detectable and/or selectable markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector. Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities. Selectable markers can be positive, negative or bifunctional. Positive selectable markers allow selection for cells carrying the marker, whereas negative selectable markers allow cells carrying the marker to be selectively eliminated. A variety of such marker genes have been described, including bifunctional (i.e., positive/negative) markers (see, e.g., WO 92/08796; and WO 94/28143). Such marker genes can provide an added measure of control that can be advantageous in gene therapy contexts. A large variety of such vectors are known in the art and are generally available.

Thus, delivery vehicles within the scope of the disclosure include, but are not limited to, isolated nucleic acid, e.g., plasmid-based vectors which may be extrachromosomally maintained, and viral vectors, e.g., recombinant adenovirus, retrovirus, lentivirus, herpesvirus, poxvirus, papilloma virus, or adeno-associated virus, including viral and non-viral vectors, or proteins, which are present in for example, nanoparticles or microparticles including liposomes, e.g., neutral or cationic liposomes, such as DOSPA/DOPE, DOGS/DOPE or DMRIE/DOPE liposomes, and/or associated with other molecules such as DNA-anti-DNA antibody-cationic lipid (DOTMA/DOPE) complexes. Delivery vehicles may be administered via any route including local administration, e.g., topical, subdermal, or subcutaneous administration.

Pharmaceutical Compositions

The disclosure provides a composition comprising, consisting essentially of, or consisting of a delivery vehicle and optionally a pharmaceutically acceptable (e.g., physiologically acceptable) carrier. When the composition consists essentially of the delivery vehicle, additional components can be included that do not materially affect the composition (e.g., adjuvants, buffers, stabilizers, anti-inflammatory agents, solubilizers, preservatives, etc.). When the composition consists of the delivery vehicle, the composition does not comprise any additional components. Any suitable carrier can be used within the context of the disclosure, and such carriers are well known in the art. The choice of carrier will be determined, in part, by the particular site to which the composition may be administered and the particular method used to administer the composition. The composition optionally can be sterile. The composition can be frozen or lyophilized for storage and reconstituted in a suitable sterile carrier prior to use. The compositions can be generated in accordance with conventional techniques described in, e.g., Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).

Suitable formulations for the composition include aqueous and non-aqueous solutions, isotonic sterile solutions, which can contain antioxidants, buffers, and bacteriostats, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. Extemporaneous solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. In one embodiment, the carrier is a buffered saline solution.

In addition, one of ordinary skill in the art will appreciate that the delivery vehicle can be present with other therapeutic or biologically active agents. For example, factors that control inflammation, such as ibuprofen or steroids, can be part of the composition to reduce swelling and inflammation associated with in vivo administration of cells having the gene therapy vector. Immune system stimulators or inhibitors, or adjuvants, e.g., interleukins, lipopolysaccharide, and double-stranded RNA, can be administered. Antibiotics, i.e., microbicides and fungicides, can be present to treat existing infection and/or reduce the risk of future infection, such as infection associated with procedures.

Injectable depot forms are made by forming microencapsulated matrices with the cells in biodegradable polymers such as polylactide-polyglycolide. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the cells in liposomes or microemulsions which are compatible with body tissue.

In certain embodiments, a formulation comprises a biocompatible polymer selected from the group consisting of polyamides, polycarbonates, polyalkylenes, polymers of acrylic and methacrylic esters, polyvinyl polymers, polyglycolides, polysiloxanes, polyurethanes and copolymers thereof, celluloses, polypropylene, polyethylenes, polystyrene, polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, poly(butic acid), poly(valeric acid), poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronic acids, polycyanoacrylates, and blends, mixtures, or copolymers thereof.

The composition can be administered in or on a device that allows controlled or sustained release, such as a sponge, biocompatible meshwork, mechanical reservoir, or mechanical implant. Implants (see, e.g., U.S. Patent No. 5,443,505), devices (see, e.g., U.S. Patent No. 4,863,457), such as an implantable device, e.g., a mechanical reservoir or an implant or a device comprised of a polymeric composition, are particularly useful for administration of the delivery vehicle. The composition also can be administered in the form of sustained-release formulations (see, e.g., U.S. Patent No. 5,378,475) comprising, for example, gel foam, hyaluronic acid, gelatin, chondroitin sulfate, a polyphosphoester, such as bis-2-hydroxyethyl-terephthalate (BHET), and/or a polylactic-glycolic acid.

Delivery of the compositions comprising the delivery vehicle may be local using devices known in the art. Delivery may also be via surgical implantation of an implanted device.

The dose of the delivery vehicle in the composition administered to the mammal will depend on a number of factors, including the size (mass) of the mammal, the extent of any sideeffects, the particular route of administration, and the like. In one embodiment, the method comprises administering a “therapeutically effective amount” of the composition comprising the delivery vehicle described herein. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. The therapeutically effective amount may vary according to factors such as the age, sex, and weight of the individual, and the ability of the delivery vehicle to elicit a desired response in the individual. The dose of a gene therapy vector in the composition required to achieve a particular therapeutic effect typically is in units of vector genome copies per cell (gc/cell) or vector genome copies/per kilogram of body weight (gc/kg). One of ordinary skill in the art can readily determine an appropriate dose range for a gene therapy vector, based on factors that are well known in the art. The therapeutically effective amount may be between 1 x IO¹⁰ genome copies to 1 x 10¹³ genome copies. The amount may be between 1 x 10¹¹ genome copies to 1 x 10¹⁴ genome copies. The amount may be between 1 x 10¹² genome copies to 1 x 10¹⁵ genome copies. The amount may be between 1 x 10¹³ genome copies to 1 x 10¹⁶ genome copies.

In one embodiment, the composition may be administered to single site of the mammal. A single administration of the composition having a gene therapy vector may result in persistent expression of a proteins such as CACNA1A in the mammal with minimal negative side effects. However, in certain cases, it may be appropriate to administer the composition multiple times during a therapeutic period to ensure sufficient exposure of the composition. For example, the composition may be administered to the mammal two or more times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times) during a period to one or more sites in the body. The present disclosure provides pharmaceutically acceptable compositions which comprise a therapeutically effective amount of a gene therapy vector comprising a nucleic acid sequence, e.g., one which encodes CACNA1 A, an isoform thereof, or a portion thereof.

In particular, administration of the gene delivery vector in accordance with the present disclosure may be a singular occurrence at one body site, multiple occurrences at one body site, a singular occurrence at multiple body site or multiple occurrences at multiple body sites, depending, for example, upon the recipient's physiological condition, the desired result, and other factors known to skilled practitioners. Both local administration, and systemic administration, are contemplated. Any direct route of administration may be employed, e.g., injection at a site in need of therapy.

One or more suitable unit dosage forms comprising delivery vehicle, which may optionally be formulated for sustained release, can be administered by a variety of routes. The formulations may, where appropriate, include the step of bringing into association the delivery vehicle with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.

The amount of the gene delivery vector(s) administered to achieve a particular outcome will vary depending on various factors including, but not limited to, the genes and promoters chosen, the condition, patient specific parameters, e.g., height, weight and age, and the desired outcome.

The gene therapy vector may conveniently be provided in the form of formulations suitable for administration, e.g., via injection. A suitable administration format may best be determined by a medical practitioner for each patient individually, according to standard procedures. Suitable pharmaceutically acceptable carriers and their formulation are described in standard formulations treatises, e.g., Remington's Pharmaceuticals Sciences. By "pharmaceutically acceptable" it is meant a carrier, diluent, excipient, and/or salt that is compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.

The gene therapy vector may be formulated in solution at neutral pH, for example, about pH 6.5 to about pH 8.5, or from about pH 7 to 8, with an excipient to bring the solution to about isotonicity, for example, 4.5% mannitol or 0.9% sodium chloride, pH buffered with art-known buffer solutions, such as sodium phosphate, that are generally regarded as safe, together with an accepted preservative such as metacresol 0.1% to 0.75%, or from 0.15% to 0.4% metacresol. Obtaining a desired isotonicity can be accomplished using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol, polyols (such as mannitol and sorbitol), or other inorganic or organic solutes. Sodium chloride is useful for buffers containing sodium ions. If desired, solutions of the above compositions can also be prepared to enhance shelf life and stability. Therapeutically useful compositions of the disclosure can be prepared by mixing the ingredients following generally accepted procedures. For example, the selected components can be mixed to produce a concentrated mixture which may then be adjusted to the final concentration and viscosity by the addition of water and/or a buffer to control pH or an additional solute to control tonicity.

The vectors for delivery to cells can be provided in a dosage form containing an amount of a vector effective in one or multiple doses. For viral vectors, the dose may be in the range of at least about 10⁷ viral particles, e.g., about 10⁹ viral particles, or about 10¹¹ viral particles. The number of viral particles added may be up to IO¹⁴ For example, when a viral expression vector is employed, about 10⁸ to about 10¹⁶ gc of viral vector can be nucleic acid or as a packaged virion. In some embodiments, about 10⁹ to about 10¹⁵ copies of viral vector, e.g., per 0.5 to 10 mL, can be employed as nucleic acid or as a packaged virion. The nucleic acids or other vectors, can be employed in dosages of at least about 0.0001 mg/kg to about 1 mg/kg, of at least about 0.001 mg/kg to about 0.5 mg/kg, at least about 0.01 mg/kg to about 0.25mg/kg or at least about 0.01 mg/kg to about 0.25 mg/kg of body weight, although other dosages may provide beneficial results. The amount will vary depending on various factors including, but not limited to, the nucleic acid or vector chosen for administration, the weight, the physical condition, the health, and/or the age of the mammal. Such factors can be readily determined by the clinician employing animal models or other test systems that are available in the art. As noted, the exact dose of cells having the gene therapy vector to be administered is determined by the attending clinician, but may be in 1 mL phosphate buffered saline. For delivery of plasmid DNA alone, or plasmid DNA in a complex with other macromolecules, the amount of DNA to be delivered to cells can vary. For example, from 0.0001 to 1 mg or more, e.g., up to 1 g, in individual or divided doses, e.g., from 0.001 to 0.5 mg, or 0.01 to 0.1 mg, of DNA.

For example, when a viral expression vector is employed, about 10⁸ to about IO⁶⁰ gc of viral vector can be employed as nucleic acid or as a packaged virion. In some embodiments, about 10⁹ to about 10¹⁵ copies of viral vector, e.g., per 0.5 to 10 mL, can be administered as nucleic acid or as a packaged virion. Alternatively, the nucleic acids or vectors, can be employed in dosages of at least about 0.0001 mg/kg to about Img/kg, of at least about 0.001 mg/kg to about 0.5 mg/kg, at least about 0.01 mg/kg to about 0.25 mg/kg or at least about 0.01 mg/kg to about 0.25 mg/kg of body weight, although other dosages may provide beneficial results.

In one embodiment, administration may be by injection or infusion using an appropriate catheter or needle. A variety of catheters may be used to achieve delivery, as is known in the art. For example, a variety of general purpose catheters, as well as modified catheters, suitable for use in the present disclosure are available from commercial suppliers.

By way of illustration, liposomes and other lipid-containing gene delivery complexes can be used to deliver one or more transgenes. The principles of the preparation and use of such complexes for gene delivery have been described in the art (see, e.g., Ledley, (1995); Miller et al., (1995); Chonn et al., (1995); Schofield et al., (1995); Brigham et al., (1993)).

Pharmaceutical formulations containing the gene delivery vectors can be prepared by procedures known in the art using well known and readily available ingredients. For example, the vector can be formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, suspensions, powders, and the like. The vectors of the disclosure can also be formulated as elixirs or solutions appropriate for parenteral administration, for instance, by intramuscular, subcutaneous or intravenous routes.

In one embodiment, the gene therapy vector may be formulated for administration, e.g., by injection, for example, bolus injection or continuous infusion via a catheter, and may include an added preservative.

These formulations can contain pharmaceutically acceptable vehicles and adjuvants which are well known in the prior art. It is possible, for example, to prepare solutions using one or more organic solvent(s) that is/are acceptable from the physiological standpoint.

The local delivery of the gene therapy vector can also be by a variety of techniques which administer the vector at or near the site of disease, e.g., using a catheter or needle. Examples of site-specific or targeted local delivery techniques are not intended to be limiting but to be illustrative of the techniques available. Examples include local delivery catheters, such as an infusion or indwelling catheter, e.g., a needle infusion catheter, shunts and stents or other implantable devices, site specific carriers, direct injection, or direct applications.

The formulations and compositions described herein may also contain other ingredients such as antimicrobial agents or preservatives.

Exemplary Particle Formulations

One or more polypeptides or polynucleotides may be present in nanoparticles or microparticles.

In one embodiment, the particles are biodegradable particles that may include or may be formed from biodegradable polymeric molecules which may include, but are not limited to polylactic acid (PLA), polyglycolic acid (PGA), co-polymers of PLA and PGA (i.e., polyactic- co-gly colic acid (PLGA)), poly-s-caprolactone (PCL), polyethylene glycol (PEG), poly(3- hydroxybutyrate), poly(p-dioxanone), polypropylene fumarate, poly(orthoesters), polyol/diketene acetals addition polymers, poly-alkyl-cyano-acrylates (PAC), poly(sebacic anhydride) (PSA), poly(carboxybiscarboxyphenoxyphenoxy hexone (PCPP) polyfbis (p- carboxypheonoxy)methane](PCPM), copolymers of PSA, PCPP and PCPM, poly(amino acids), poly(pseudo amino acids), polyphosphazenes, derivatives of poly[(dichloro)phosphazenes] and poly[(organo)phosphazenes], poly-hydroxybutyric acid, or S-caproic acid, elastin, or gelatin. (See, e.g., Kumari et al., Colloids and Surfaces B: Biointerfaces 75 (2010) 1-18; and U.S. Patent Nos. 6,913,767; 6,884,435; 6,565,777; 6,534,092; 6,528,087; 6,379,704; 6,309,569; 6,264,987; 6,210,707; 6,090,925; 6,022,564; 5,981,719; 5,871,747; 5,723,269; 5,603,960; and 5,578,709; and U.S. Published Application No. 2007/0081972; and International Application Publication Nos. WO 2012/115806; and WO 2012/054425; the contents of which are incorporated herein by reference in their entireties).

The biodegradable particles may be prepared by methods known in the art. The size of the particles (e.g., mean effective diameter) may be assessed by known methods in the art, which may include but are not limited to transmission electron microscopy (TEM), scanning electron microscopy (SEM), Atomic Force Microscopy (AFM), Photon Correlation Spectroscopy (PCS), Nanoparticle Surface Area Monitor (NS AM), Condensation Particle Counter (CPC), Differential Mobility Analyzer (DMA), Scanning Mobility Particle Sizer (SMPS), Nanoparticle Tracking Analysis (NTA), X-Ray Diffraction (XRD), Aerosol Time of Flight Mass Spectroscopy (ATFMS), and Aerosol Particle Mass Analyzer (APM).

In one embodiment, a particle comprises polymers including but not limited to poly(lactic- co-glycolic acid) (PLGA), polylactic acid (PLA), linear and/or branched PEI with differing molecular weights (e.g., 2, 22 and 25 kDa), dendrimers such as polyamidoamine (PAMAM) and polymethoacrylates; lipids including but not limited to cationic liposomes, cationic emulsions, DOTAP, DOTMA, DMRIE, DOSPA, distearoylphosphatidylcholine (DSPC), DOPE, or DC- cholesterol; peptide based vectors including but not limited to Poly-L-lysine or protamine; or poly(P-amino ester), chitosan, PEI-polyethylene glycol, PEI-mannose-dextrose, DOTAP- cholesterol or RNAiMAX.

In one embodiment, the delivery vehicle may be a glycopolymer-based delivery vehicle, poly(glycoamidoamine)s (PGAAs), that have the ability to complex with various polynucleotide types and form nanoparticles. These materials are created by polymerizing the methylester or lactone derivatives of various carbohydrates (D-glucarate (D), /7/c.w-galactarate (G), D-mannarate (M), and L-tartarate (T)) with a series of oligoethyleneamine monomers (containing between 1-4 ethylenamines (Liu and Reineke, 2006)). A subset composed of these carbohydrates and four ethyleneamines in the polymer repeat units yielded exceptional delivery efficiency. In one embodiment, the delivery vehicle may comprise polyethyleneimine (PEI), Polyamidoamine (PAMAM), PEI-PEG, PEI-PEG-mannose, dextran-PEI, OVA conjugate, PLGA microparticles, or PLGA microparticles coated with PAMAM, or any combination thereof. The disclosed cationic polymer may include, but are not limited to, polyamidoamine (PAMAM) dendrimers. Polyamidoamine dendrimers suitable for preparing the presently disclosed nanoparticles may include 3rd-, 4th-, 5th-, or at least 6th-generation dendrimers.

In one embodiment, the delivery vehicle may comprise a lipid, e.g., 7V-[l-(2,3- di oleoyloxy )propel]-7V,7V,7V-trimethylammonium (DOTMA), 2,3 -dioleyloxy-V-[2-spermine carboxamide] ethyl-7V,7V-dimethyl-l-propanammonium trifluoracetate (DOSPA, Lipofectamine); l,2-dioleoyl-3-trimethylammonium-propane (DOTAP); 7V-[l-(2,3-dimyristloxy) propyl]; N,N- dimethyl-V-(2-hydroxyethyl) ammonium bromide (DMRIE), 3-P-[7V-(7V,V- dimethylaminoethane) carbamoyl] cholesterol (DC-Chol); dioctadecyl amidoglyceryl spermine (DOGS, Transfectam); or imethyldioctadeclyammonium bromide (DDAB). The positively charged hydrophilic head group of cationic lipids usually consists of monoamine such as tertiary and quaternary amines, polyamine, amidinium, or guanidinium group. A series of pyridinium lipids have been developed (Zhu et al., 2008; van der Woude et al., 1997; Hies et al., 2004). In addition to pyridinium cationic lipids, other types of heterocyclic head group include imidazole, piperizine and amino acid. The main function of cationic head groups is to condense negatively charged nucleic acids by means of electrostatic interaction to slightly positively charged nanoparticles, leading to enhanced cellular uptake and endosomal escape.

Lipids having two linear fatty acid chains, such as DOTMA, DOTAP and SAINT-2, or DODAC, may be employed as a delivery vehicle, as well as tetraalkyl lipid chain surfactant, the dimer of N,N-dioleyl-NA- dimethylammonium chloride (DODAC). All the /ra/z.s-orientated lipids regardless of their hydrophobic chain lengths (Ci6:i, Ci8:i and C20:i) appear to enhance the transfection efficiency compared with their cv.s-orientated counterparts.

The structures of polymers useful as a delivery vehicle include but are not limited to linear polymers such as chitosan and linear poly(ethyleneimine), branched polymers such as branch poly(ethyleneimine) (PEI), circle-like polymers such as cyclodextrin, network (crosslinked) type polymers such as crosslinked poly(amino acid) (PAA), and dendrimers. Dendrimers consist of a central core molecule, from which several highly branched arms 'grow' to form a tree-like structure with a manner of symmetry or asymmetry. Examples of dendrimers include polyamidoamine (PAMAM) and polypropylenimine (PPI) dendrimers.

DOPE and cholesterol are commonly used neutral co-lipids for preparing liposomes. Branched PELcholesterol water-soluble lipopolymer conjugates self-assemble into cationic micelles. Pluronic (pol oxamer), a non-ionic polymer and SP1017, which is the combination of Pluronics L61 and F127, may also be used.

In some embodiments, a biocompatible polymeric material is derived from a biodegradable polymeric such as collagen, e.g., hydroxylated collagen, fibrin, polylactic- polyglycolic acid, or a polyanhydride. Other examples include, without limitation, any biocompatible polymer, whether hydrophilic, hydrophobic, or amphiphilic, such as ethylene vinyl acetate copolymer (EVA), polymethyl methacrylate, polyamides, polycarbonates, polyesters, polyethylene, polypropylenes, polystyrenes, polyvinyl chloride, polytetrafluoroethylene, N- isopropyl acrylamide copolymers, poly(ethylene oxide)/poly(propylene oxide) block copolymers, poly(ethylene glycol)/poly(D,L-lactide-co-glycolide) block copolymers, polyglycolide, polylactides (PLLA or PDLA), poly(caprolactone) (PCL), or poly(dioxanone) (PPS).

In another embodiment, the biocompatible material includes polyethyleneterephalate, polytetrafluoroethylene, copolymer of polyethylene oxide and polypropylene oxide, a combination of polygly colic acid and polyhydroxyalkanoate, gelatin, alginate, poly-3 - hydroxybutyrate, poly-4-hydroxybutyrate, and polyhydroxyoctanoate, and polyacrylonitrilepolyvinylchlorides.

In one embodiment, the following polymers may be employed, e.g., natural polymers such as starch, chitin, glycosaminoglycans, e.g., hyaluronic acid, dermatan sulfate and chrondrotin sulfate, and microbial polyesters, e.g., hydroxyalkanoates such as hydroxyvalerate and hydroxybutyrate copolymers, and synthetic polymers, e.g., poly(orthoesters) and polyanhydrides, and including homo and copolymers of glycolide and lactides (e.g., poly(L-lactide, poly(L-lactide- co-D,L-lactide), poly(L-lactide-co-glycolide, polyglycolide and poly(D,L-lactide), pol(D,L- lactide-coglycolide), poly(lactic acid colysine) and polycaprolactone.

In one embodiment, the biocompatible material is derived from isolated extracellular matrix (ECM). ECM may be isolated from endothelial layers of various cell populations, tissues and/or organs, e.g., any organ or tissue source including the dermis of the skin, liver, alimentary, respiratory, intestinal, urinary or genital tracks of a warm-blooded vertebrate. ECM employed in the invention may be from a combination of sources. Isolated ECM may be prepared as a sheet, in particulate form, gel form and the like.

A biocompatible scaffold polymer may comprise silk, elastin, chitin, chitosan, polyphydroxy acid), poly(anhydrides), or poly (orthoesters). More particularly, the biocompatible polymer may be formed polyethylene glycol, poly(lactic acid), poly(glycolic acid), copolymers of lactic and glycolic acid, copolymers of lactic and glycolic acid with polyethylene glycol, poly(E- caprolactone), poly (3 -hydroxybutyrate), poly(p-dioxanone), polypropylene fumarate, poly(orthoesters), polyol/diketene acetals addition polymers, poly(sebacic anhydride) (PSA), poly(carboxybiscarboxyphenoxyphenoxy hexone (PCPP) polyfbis (p-carboxypheonoxy) methane] (PCPM), copolymers of SA, CPP and CPM, poly(amino acids), poly(pseudo amino acids), polyphosphazenes, derivatives of poly[(dichloro)phosphazenes] or polyf(organo) phosphazenes], poly-hydroxybutyric acid, or S-caproic acid, polylactide-co-glycolide, polylactic acid, polyethylene glycol, cellulose, oxidized cellulose, alginate, gelatin or derivatives thereof.

Thus, the polymer may be formed of any of a wide range of materials including polymers, including naturally occurring polymers, synthetic polymers, or a combination thereof. In one embodiment, the scaffold comprises biodegradable polymers. In one embodiment, a naturally occurring biodegradable polymer may be modified to provide for a synthetic biodegradable polymer derived from the naturally occurring polymer. In one embodiment, the polymer is a poly(lactic acid) ("PLA") or poly(lactic-co-glycolic acid) ("PLGA"). In one embodiment, the scaffold polymer includes but is not limited to alginate, chitosan, poly(2- hydroxyethylmethacrylate), xyloglucan, co-polymers of 2-methacryloyloxyethyl phosphorylcholine, poly(vinyl alcohol), silicone, hydrophobic polyesters and hydrophilic polyester, poly(lactide-co-glycolide), N-isoproyl acrylamide copolymers, poly(ethylene oxide)/poly(propylene oxide), polylactic acid, poly(orthoesters), polyanhydrides, polyurethanes, copolymers of 2-hydroxyethylmethacrylate and sodium methacrylate, phosphorylcholine, cyclodextrins, polysulfone and polyvinylpyrrolidine, starch, poly-D,L-lactic acid-para- dioxanone-polyethylene glycol block copolymer, polypropylene, poly(ethylene terephthalate), poly(tetrafluoroethylene), poly-epsilon-caprolactone, or crosslinked chitosan.

In one embodiment, the microparticles have a diameter of about 1 to about 100 microns. In one embodiment, the diameter is about 1 to about 15 microns. In one embodiment, the diameter is about 5 to about 10 microns. In one embodiment, the diameter is about 15 to about 50 microns. In one embodiment, the diameter is about 20 to about 50 microns. In one embodiment, the diameter is about 100 to about 150 microns. In one embodiment, the diameter is about 500 to about 750 microns. In one embodiment, the diameter is about 150 to about 500 microns. In one embodiment, the diameter is about 200 to about 500 microns.

In one embodiment, the nanoparticles have a diameter of about 1 to about 100 nm. In one embodiment, the diameter is about 1 to about 15 nm. In one embodiment, the diameter is about 5 to about 10 nm. In one embodiment, the diameter is about 15 to about 50 nm. In one embodiment, the diameter is about 20 to about 50 nm. In one embodiment, the diameter is about 100 to about 150 nm. In one embodiment, the diameter is about 500 to about 750 nm. In one embodiment, the diameter is about 150 to about 500 nm. In one embodiment, the diameter is about 200 to about 500 nm.

Exemplary Formulations and Dosages

The delivery vehicles can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, e.g., parenterally, or by intravenous, intramuscular, topical or subcutaneous routes.

In one embodiment, a sustained release formulation comprising polypeptides or portions thereof, or vectors having nucleic acid encoding the polypeptide or portion thereof, may be administered by infusion or injection. Solutions of the polypeptides or portion thereof, or nucleic acid encoding the polypeptide or portion thereof, or salts thereof, can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion may include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it may be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active agent in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

Useful solid carriers may include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as antimicrobial agents can be added to optimize the properties for a given use. Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

The amount of the composition required for use alone or with other agents will vary with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician.

The polypeptides or portions thereof, or vectors having nucleic acid encoding the polypeptide or portion thereof, may be conveniently administered in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, or conveniently 50 to 500 mg of active ingredient per unit dosage form.

A suitable dose may be in the range of from about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of body weight, such as 3 to about 50 mg per kilogram body weight, for example in the range of 6 to 90 mg/kg, e.g., in the range of 15 to 60 mg/kg.

Compositions Having Viral Vectors and Routes of Delivery

Any route of administration may be employed for viral vectors so long as that route and the amount administered are prophylactically or therapeutically useful.

In vivo administration of the components, e.g., delivered in a viral vector such as an Ad vector(s), and compositions containing them, can be accomplished by any suitable method and technique presently or prospectively known to those skilled in the art. The subject polynucleotides or polypeptides can be formulated in a physiologically- or pharmaceutically-acceptable form and administered by any suitable route known in the art including, for example, oral, nasal, rectal, transdermal, vaginal, and parenteral routes of administration. As used herein, the term parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, subretinal, intracochlear, intrathecal, and intracisternal administration, such as by injection.

Administration of the compositions can be a single administration, or at continuous or distinct intervals as can be readily determined by a person skilled in the art. In one embodiment, a polynucleotide component is stably incorporated into the genome of a person or an animal in need of treatment. Methods for providing gene therapy are well known in the art.

The compositions can also be administered utilizing liposome and nano-technology, slow release capsules, implantable pumps, and biodegradable containers, and orally or intestinally administered intact plant cells expressing the therapeutic product. These delivery methods can, advantageously, provide a uniform dosage over an extended period of time.

Suitable dose ranges for viral vectors are generally about 10³ to 10¹⁵ infectious units of viral vector per microliter delivered in 1 to 3000 microliters of single injection volume. For instance, viral genomes or infectious units of vector per micro liter would generally contain about 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, IO¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, or 10¹⁷ viral genomes or infectious units of viral vector delivered in about 10, 50, 100, 200, 500, 1000, or 2000 microliters. It should be understood that the aforementioned dosage is merely an exemplary dosage and those of skill in the art will understand that this dosage may be varied. Effective doses may be extrapolated from dose-responsive curves derived from in vitro or in vivo test systems.

In one embodiment, suitable dose ranges are generally about 10³ to 10¹⁵ infectious units of viral vector per microliter delivered in, for example, 1, 2, 5, 10, 25, 50, 75or 100 or more milliliters, e.g.,1 to 10,000 milliliters or 0.5 to 15 milliliters of single injection volume. For instance, viral genomes or infectious units of vector per microliter would generally contain about 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, or 10¹⁴ viral genomes or infectious units of viral vector. In one embodiment, suitable dose ranges, generally about 10³ to 10¹⁵ infectious units of viral vector per microliter delivered in, for example, 1, 2, 5, 10, 25, 50, 75 or 100 or more milliliters, e.g., 1 to 10,000 milliliters or 0.5 to 15 milliliters. For instance, viral genomes or infectious units of vector per microliter would generally contain about 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, or 10¹⁷ viral genomes or infectious units of viral vector, e.g., at least 1.2 x 10¹¹ genomes or infectious units, for instance at least 2 x 10¹¹ up to about 2 x 10¹² genomes or infectious units or about 1 x 10¹³ to about 5 x 10¹⁶ genomes or infectious units.

Administration of viral vectors in accordance with the present disclosure can be achieved by direct injection of the composition or by the use of infusion pumps. For injection, the composition can be formulated in liquid solutions, e.g., in physiologically compatible buffers such as Hank's solution, Ringer's solution or phosphate buffer.

In one embodiment, the viral vector(s) may be administered by any route including parenterally. In one embodiment, the viral vector(s) may be administered by subretinal, intracochlear, subcutaneous, intramuscular, or intravenous injection, orally, intrathecally, or intracranially, or by sustained release, e.g., using a subcutaneous implant. The viral vector(s) may be dissolved or dispersed in a liquid carrier vehicle. For parenteral administration, the active material may be suitably admixed with an acceptable vehicle, e.g., of the vegetable oil variety such as peanut oil, cottonseed oil and the like. Other parenteral vehicles such as organic compositions using solketal, glycerol, formal, and aqueous parenteral formulations may also be used. For parenteral application by injection, the viral vector(s) may comprise an aqueous solution of a water soluble pharmaceutically acceptable salt of the active acids according to the invention, desirably in a concentration of 0.01-10%, and optionally also a stabilizing agent and/or buffer substances in aqueous solution. Dosage units of the solution may advantageously be enclosed in ampules.

The viral vector(s) may be in the form of an injectable unit dose. Examples of carriers or diluents usable for preparing such injectable doses include diluents such as water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxyisostearyl alcohol and polyoxyethylene sorbitan fatty acid esters, pH adjusting agents or buffers such as sodium citrate, sodium acetate and sodium phosphate, stabilizers such as sodium pyrosulfite, EDTA, thioglycolic acid and thiolactic acid, isotonic agents such as sodium chloride and glucose, local anesthetics such as procaine hydrochloride and lidocaine hydrochloride. Furthermore, usual solubilizing agents and analgesics may be added. Injections can be prepared by adding such carriers to the enzyme or other active, following procedures well known to those of skill in the art. A thorough discussion of pharmaceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991). The pharmaceutically acceptable formulations can easily be suspended in aqueous vehicles and introduced through conventional hypodermic needles or using infusion pumps. Prior to introduction, the formulations can be sterilized, e.g., using filters.

When the viral vector(s) is administered in the form of a subcutaneous implant, the compound is suspended or dissolved in a slowly dispersed material known to those skilled in the art, or administered in a device which slowly releases the active material through the use of a constant driving force such as an osmotic pump. In such cases, administration over an extended period of time is possible.

The dosage at which the viral vector(s) is administered may vary within a wide range and will depend on various factors such as the severity of the disease, the age of the patient, etc., and may have to be individually adjusted. Compositions described herein may be employed in combination with another medicament. The compositions can appear in conventional forms, for example, aerosols, solutions, suspensions, or topical applications, or in lyophilized form. Typical compositions include the viral vector(s) and a pharmaceutically acceptable excipient which can be a carrier or a diluent. For example, the active agent(s) may be mixed with a carrier, or diluted by a carrier, or enclosed within a carrier. When the active agent is mixed with a carrier, or when the carrier serves as a diluent, it can be solid, semi-solid, or liquid material that acts as a vehicle, excipient, or medium for the active agent. Some examples of suitable carriers are water, salt solutions, alcohols, polyethylene glycols, polyhydroxyethoxylated castor oil, peanut oil, olive oil, gelatin, lactose, terra alba, sucrose, dextrin, magnesium carbonate, sugar, cyclodextrin, amylose, magnesium stearate, talc, gelatin, agar, pectin, acacia, stearic acid or lower alkyl ethers of cellulose, silicic acid, fatty acids, fatty acid amines, fatty acid monoglycerides and diglycerides, pentaerythritol fatty acid esters, polyoxyethylene, hydroxymethylcellulose and polyvinylpyrrolidone. Similarly, the carrier or diluent can include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.

The formulations can be mixed with auxiliary agents which do not deleteriously react with the vector(s). Such additives can include wetting agents, emulsifying and suspending agents, salt for influencing osmotic pressure, buffers and/or coloring substances preserving agents, sweetening agents or flavoring agents. The compositions can also be sterilized if desired.

If a liquid carrier is used, the preparation can be in the form of a liquid such as an aqueous liquid suspension or solution. Acceptable solvents or vehicles include sterilized water, Ringer's solution, or an isotonic aqueous saline solution.

The viral vector(s) may be provided as a powder suitable for reconstitution with an appropriate solution as described above. Examples of these include, but are not limited to, freeze dried, rotary dried or spray dried powders, amorphous powders, granules, precipitates, or particulates. The composition can optionally contain stabilizers, pH modifiers, surfactants, bioavailability modifiers and combinations of these. A unit dosage form can be in individual containers or in multi-dose containers.

In one embodiment, the preparation can contain an agent, dissolved or suspended in a liquid carrier, such as an aqueous carrier, for aerosol application. The carrier can contain additives such as solubilizing agents, e.g., propylene glycol, surfactants, absorption enhancers such as lecithin (phosphatidylcholine) or cyclodextrin, or preservatives such as parabens.

Exemplary Genes and Gene Products for Delivery

Exemplary genes for delivery to various organs include but are not limited to genes encoding ABCA4, ALFY, CEP290, USH2A, MY07A, PCDH15, CACNA1F, CDH23, OTOF, DYSF, ALMS1, DMD, CACNA1B, CACNA1E, RIMS, MUNC13, SCN2A, CACNA1D, CACNA1H, and the like. In one embodiment, the gene encodes CACNA1A (voltage-dependent P/Q-type calcium channel subunit alpha-lA isoform 1 (Homo sapiens)) having NCBI Reference Sequence: NP_000059.3, e.g., mart gdempa ryggggsgaa agvvvgsggg rgaggs rqgg qpgaqrmykq smaqrartma lynpipvrqn cltvnrsl fl f sednvvrky akkitewppf eymilatiia ncivlaleqh Ipdddktpms erlddtepyf igi f cfeagi kiialgf afh kgsylrngwn vmdfvvvltg ilatvgtefd Irtlravrvl rplklvsgip slqvvlksim kamipllqig lll ffaili f aiiglef ymg kfhttcf eeg tddiqgespa pegteepart cpngtkcqpy wegpnngitq fdnil favlt vf qcitmegw tdllynsnda sgntwnwlyf ipliiigs f f mlnlvlgvl s gef akererv enrraf Iklr rqqqiereln gymewis kae evilaedetd geqrhpfdga Irrttikks k tdllnpeeae dqladiasvg spf arasiks aklenstf fh kkerrmrf yi rrmvktqaf y wtvlslvaln tlcvaivhyn qpewlsdf ly yaefi flgl f msemf ikmyg Igtrpyfhs s fncfdcgvii gsi feviwav ikpgts f gis vlralrllri f kvtkywas l rnlvvsllns mksiisll fl I fl fivvfal Igmql fggqf nfdegtpptn fdtfpaaimt vf qiltgedw nevmydgiks qggvqggmvf siyfivltl f gnytllnvf 1 aiavdnlana qeltkveade qeeeeaanqk lalqkakeva evsplsaanm siavkeqqkn qkpaksvweq rtsemrkqnl las realyne mdpderwkaa ytrhlrpdmk thldrplvvd pqenrnnntn ks raaeptvd qrlgqqraed f Irkqaryhd rardpsgsag Idarrpwags qeaels regp ygresdhhar egsleqpgfw egeaergkag dphrrhvhrq ggs res rsgs prtgadgehr rhrahrrpge egpedkaerr arhregs rpa rggegegegp dggerrrrhr hgapatyegd arredkerrh rrrkenqgsg vpvsgpnlst trpiqqdlgr qdpplaedid nmknnklata esaaphgslg haglpqspak mgnstdpgpm laipamatnp qnaasrrtpn npgnpsnpgp pktpensliv tnpsgtqtns aktarkpdht tvdippaepp plnhtvvqvn knanpdplpk keeekkeeee ddrgedgpkp mppys smfil sttnplrrlc hyilnlryf e mcilmviams sialaaedpv qpnaprnnvl ryfdyvf tgv f tf emvikmi dlglvlhqga yf rdlwnild f ivvsgalva faftgns kgk dintikslrv Irvlrplkti krlpklkavf dcvvnslknv fnilivyml f mfi favvavq I f kgkf fhet des kefekdc rgkyllyekn evkardrewk kyefhydnvl walltl ftvs tgegwpqvlk hsvdatf enq gpspgyrmem si fyvvyfvv fpf f fvni fv aliiitf qeq gdkmmeeysl ekneracidf aisakpltrh mpqnkqs f qy rmwqfvvspp f eytimamia Intivlmmkf ygasvayena Irvfnivf ts I f slecvlkv maf gilnyf r dawni fdfvt vlgsitdilv tef gnpnnf i nls flrl fra arlikllrqg ytirillwtf vqs f kalpyv clliaml f fi yaiigmqvf g nigidveded sdedef qite hnnf rtf f qa Imll f rsatg eawhnimlsc Isgkpcdkns giltreegne f ayf yfvs f i f les f Imlnl fvavimdnf e yltrds silg phhldeyvrv waeydpaawg rmpyldmyqm Irhmspplgl gkkeparvay krllrmdlpv addntvhfns tlmalirtal dikiakggad kqqmdaelrk emmaiwpnls qktldllvtp hkstdltvgk iyaammimey yrqs kakklq amreeqdrtp Imf qrmepps ptqeggpgqn alpstqldpg galmahesgl kespswvtqr aqemf qktgt wspeqgpptd mpnsqpnsqs vemremgrdg ysdsehylpm egqgraasmp rlpaenqrrr grprgnnlst isdtspmkrs asvlgpkarr Iddyslervp peenqrhhqr rrdrshrase rslgrytdvd tglgtdlsmt tqsgdlps ke rdqergrpkd rkhrqhhhhh hhhhhppppd kdryaqerpd hgrarardqr ws rspsegre hmahrq

(SEQ ID NO: 1), a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto. In one embodiment, the gene encodes ATM (serine-protein kinase ATM isoform a (Homo sapiens)), such as NCBI Reference Sequence: NP_000042.3 having: mslvlndlli ccrqlehdra terkkevekf krlirdpeti khldrhsdsk qgkylnwdav f rf Iqkyiqk eteclriakp nvsastqasr qkkmqeissl vkyf ikcanr raprlkcqel Inyimdtvkd ssngaiygad csnillkdil svrkywceis qqqwlelf sv yf rlylkpsq dvhrvlvari ihavtkgccs qtdglnskfl df f skaiqca rqeksssgln hilaaltif 1 ktlavnf rir vcelgdeilp tllyiwtqhr Indslkevii elfqlqiyih hpkgaktqek gayestkwrs ilynlydllv neishigsrg kyssgf rnia vkenlielma dichqvfned trsleisqsy tttqressdy svpckrkkie Igwevikdhl qksqndfdlv pwlqiatqli skypaslpnc elspllmils qllpqqrhge rtpyvlrclt evalcqdkrs nlessqksdl Iklwnkiwci tf rgisseqi qaenf gllga iiqgslvevd refwklftgs acrpscpavc cltlalttsi vpgtvkmgie qnmcevnrs f slkesimkwl Ifyqlegdle nstevppilh snfphlvlek ilvsltmknc kaamnf f qsv pecehhqkdk eels f sevee Iflqttfdkm df Itivrecg iekhqssigf svhqnlkesl drcllglseq llnnysseit nsetlvrcs r llvgvlgcyc ymgviaeeea ykselfqkak slmqcagesi tlf knktnee f rigslrnmm qlctrclsnc tkkspnkias gfflrlltsk Imndiadick slas f ikkpf drgevesmed dtngnlmeve dqssmnlfnd ypdssvsdan epgesqstig ainplaeeyl skqdllfldm Ikf Iclcvtt aqtntvs f ra adirrkllml idsstleptk slhlhmylml Ikelpgeeyp Ipmedvlell kplsnvcsly rrdqdvckti Inhvlhvvkn Igqsnmdsen trdaqgqf It vigafwhltk erkyif svrm alvnclktll eadpyskwai Invmgkdfpv nevf tqf lad nhhqvrmlaa esinrlfqdt kgdssrllka Iplklqqtaf enaylkaqeg mremshsaen petldeiynr ksvlltliav vlscspicek qalfalcksv kenglephlv kkvlekvset f gyrrledfm ashidyl vie wlnlqdteyn Issfpfilln ytniedf yrs cykvliphlv irshfdevks ianqiqedwk slltdcfpki Ivnilpyf ay egtrdsgmaq qretatkvyd mlksenllgk qidhlfisnl peivvellmt lhepanssas qstdlcdf sg dldpapnpph fpshvikatf ayisnchktk Iksileilsk spdsyqkill aiceqaaetn nvykkhrilk iyhlfvslll kdiksglgga wafvlrdviy tlihyinqrp scimdvslrs f slccdllsq vcqtavtyck dalenhlhvi vgtliplvye qvevqkqvld llkylvidnk dnenlyitik lldpfpdhvv f kdlritqqk ikysrgpf si leeinhf Isv svydalpltr leglkdlrrq lelhkdqmvd imrasqdnpq dgimvklvvn llqlskmain htgekevlea vgsclgevgp idf stiaiqh skdasytkal klfedkelqw tf imltylnn tlvedcvkvr saavtclkni latktghs fw eiykmttdpm laylqpf rts rkkf levprf dkenpf egld dinlwiplse nhdiwiktlt caf Idsggtk ceilqllkpm cevktdf cqt vlpylihdil Iqdtneswrn llsthvqgf f tsclrhf sqt srsttpanld sesehf f rcc Idkksqrtml avvdymrrqk rpssgtifnd afwldlnyle vakvaqscaa hf tallyaei yadkksmddq ekrslaf eeg sqsttissls ekskeetgis Iqdllleiyr sigepdslyg cgggkml qpi trlrtyehea mwgkalvtyd letaipsstr qagiiqalqn Iglchilsvy Ikgldyenkd wcpeleelhy qaawrnmqwd hctsvskeve gtsyheslyn alqslrdref stf yeslkya rvkeveemck rslesvysly ptlsrlqaig elesigelf s rsvthrqlse vyikwqkhsq llkdsdf s f q epimalrtvi leilmekemd nsqrecikdi Itkhlvelsi lartf kntql peraifqikq ynsvscgvse wqleeaqvfw akkeqslals ilkqmikkld ascaannpsl kltyteclrv cgnwlaetcl enpavimqty lekavevagn ydgessdelr ngkmkaf Isl arf sdtqyqr ienymkssef enkqallkra keevgllreh kiqtnrytvk vqreleldel alralkedrk rf Ickaveny incllsgeeh dmwvf rlcsl wlensgvsev ngmmkrdgmk iptykf Iplm yqlaarmgtk mmgglgfhev Innlisrism dhphhtlfii lalananrde f Itkpevarr sritknvpkq ssqldedrte aanriictir srrpqmvrsv ealcdayiil anldatqwkt qrkginipad qpitklknle dvvvptmeik vdhtgeygnl vtiqs f kaef rlaggvnlpk iidcvgsdgk errqlvkgrd dlrqdavmqq vf qmcntllq rntetrkrkl tictykvvpl sqrsgvlewc tgtvpigef 1 vnnedgahkr yrpndf saf q cqkkmmevqk ks f eekyevf mdvcqnf qpv f ryf cmekf 1 dpaiwf ekrl aytrsvatss ivgyilglgd rhvqniline qsaelvhidl gvaf eqgkil ptpetvpf rl trdivdgmgi tgvegvf rrc cektmevmrn sqetlltive vllydplfdw tmnplkalyl qqrpedetel hptlnaddqe ckrnlsdidq s fnkvaervl mrlqeklkgv eegtvlsvgg qvnlliqqai dpknlsrlfp gwkawv

(SEQ ID NO:2), a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.

In one embodiment, the gene encodes huntingtin isoform I (Homo sapiens) such as one having NCBI Reference Sequence: NP 001375421.1: matleklmka f eslks f qqq qqqqqqqqqq qqqqqqqqpp pppppppppq Ipqpppqaqp llpqpqpppp ppppppgpav aeeplhrpkk elsatkkdrv nhclticeni vaqsvrnspe f qkllgiame Ifllcsddae sdvrmvadec Inkvikalmd snlprlqlel ykeikkngap rslraalwrf aelahlvrpq kcrpylvnll pcltrtskrp eesvqetlaa avpkimas f g nf andneikv llkaf ianlk sssptirrta agsavsicqh srrtqyfysw llnvllgllv pvedehstll ilgvlltlry Ivpllqqqvk dtslkgs f gv trkemevsps aeqlvqvyel tlhhtqhqdh nvvtgalell qqlf rtpppe llqtltavgg igqltaakee sggrsrsgsi veliagggss cspvlsrkqk gkvllgeeea leddsesrsd vsssaltasv kdeisgelaa ssgvstpgsa ghdiiteqpr sqhtlqadsv dlascdltss atdgdeedil shsssqvsav psdpamdlnd gtqasspisd ssqtttegpd savtpsdsse ivldgtdnqy Iglqigqpqd edeeatgilp deaseaf rns smalqqahll knmshcrqps dssvdkfvlr deatepgdqe nkpcrikgdi gqstdddsap Ivhcvrllsa s f lltggknv Ivpdrdvrvs vkalalscvg aavalhpes f f sklykvpld tteypeeqyv sdilnyidhg dpqvrgatai Icgtlicsil srsrfhvgdw mgtirtltgn tf sladcipl Irktlkdess vtcklactav rncvmslcs s syselglqli idvltlrnss ywlvrtelle tlaeidf rlv s f leakaenl hrgahhytgl Iklqervlnn vvihllgded prvrhvaaas lirlvpklfy kcdqgqadpv vavardqssv ylkllmhetq ppshf svsti triyrgynll psitdvtmen nlsrviaavs helitsttra Itf gccealc llstafpvci wslgwhcgvp plsasdesrk sctvgmatmi Itllssawfp Idlsahqdal ilagnllaas apkslrsswa seeeanpaat kqeevwpalg dralvpmveq If shllkvin icahvlddva pgpaikaalp sltnppslsp irrkgkekep geqasvplsp kkgseasaas rqsdtsgpvt tskssslgsf yhlpsylklh dvlkathany kvtldlqnst ekf ggf Irsa Idvlsqilel atlqdigkcv eeilgylksc f srepmmatv cvqqllktl f gtnlasqfdg Issnpsksqg raqrlgsssv rpglyhycfm apythf tqal adaslrnmvq aeqendtsgw fdvlqkvstq Iktnltsvtk nradknaihn hirlfeplvi kalkqytttt cvqlqkqvld llaqlvqlrv nyclldsdqv f igfvlkqf e yievgqf res eaiipnif f f Ivllsyeryh skqiigipki iqlcdgimas grkavthaip alqpivhdlf vlrgtnkada gkeletqkev vvsmllrliq yhqvlemf il vlqqchkene dkwkrlsrqi adiilpmlak qqmhidshea Igvlntlfei lapsslrpvd mllrsmfvtp ntmasvstvq Iwisgilail rvlisqsted ivlsriqels f spylisctv inrlrdgdst stleehsegk qiknlpeetf srfllqlvgi lledivtkql kvemseqqht f ycqelgtll mclihif ksg mf rritaaat rlf rsdgcgg s f ytldslnl rarsmitthp alvllwcqil llvnhtdyrw waevqqtpkr hsls stklls pqmsgeeeds dlaaklgmcn reivrrgali I f cdyvcqnl hdsehltwli vnhiqdlisl sheppvqdf i savhrnsaas gl fiqaiqs r cenlstptml kktlqclegi hlsqsgavlt lyvdrllctp f rvlarmvdi lacrrvemll aanlqs smaq Ipmeelnriq eylqs sglaq rhqrlyslld rf rlstmqds Ispsppvs sh pldgdghvsl etvspdkdwy vhlvksqcwt rsdsallega elvnripaed mnafmmnsef nlsllapcls Igmseisggq ksal feaare vtlarvsgtv qqlpavhhvf qpelpaepaa yws klndl fg daalyqslpt laralaqylv vvs klpshlh Ippekekdiv kfvvatleal swhliheqip Isldlqagld ccclalqlpg Iwsvvs stef vthacsliyc vhf ileavav qpgeqllspe rrtntpkai s eeeeevdpnt qnpkyitaac emvaemvesl qsvlalghkr nsgvpaf Itp llrniiisla rlplvnsytr vpplvwklgw spkpggdf gt afpeipvef 1 qekevf kef i yrintlgwts rtqf eetwat llgvlvtqpl vmeqeesppe edtertqinv lavqaitslv Isamtvpvag npavs cleqq prnkplkald trf grklsii rgiveqeiqa mvs kreniat hhlyqawdpv pslspattga lisheklllq inperelgsm syklgqvsih svwlgnsitp Ireeewdeee eeeadapaps spptspvns r khragvdihs csqf llelys rwilps s sar rtpailisev vrsllvvsdl f ternqf elm yvtltelrrv hpsedeilaq ylvpatckaa avlgmdkava epvs rllest Irs shlps rv galhgvlyvl ecdllddtak qlipvisdyl Isnlkgiahc vnihsqqhvl vmcataf yli enypldvgpe f sasiiqmcg vmlsgseest psiiyhcal r glerlllseq Is rldaeslv klsvdrvnvh sphramaalg Imltcmytgk ekvspgrtsd pnpaapdses vivamervsv I fdrirkgfp cearvvaril pqf Iddf fpp qdimnkvige f Isnqqpypq fmatvvykvf qtlhstgqs s mvrdwvml s 1 snf tqrapva matwslscf f vsastspwva ailphvis rm gkleqvdvnl f clvatdf yr hqieeeldrr af qsvlevva apgspyhrll tclrnvhkvt tc

(SEQ ID NO:3), a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.

In one embodiment, the gene encodes ataxin-1 isoform ATXN1 (Homo sapiens) having NCBI Reference Sequence: NP ,001121636 I : mksnqersne clppkkreip ats rs seeka ptlpsdnhrv egtawlpgnp ggrghgggrh gpagtsvelg Iqqgiglhka Istgldyspp saprsvpvat tlpaayatpq pgtpvspvqy ahlphtf qf i gs sqysgtya s f ipsqlipp tanpvtsava saagattpsq rsqleaystl lanmgslsqt pghkaeqqqq qqqqqqqqhq hqqqqqqqqq qqqqqhls ra pglitpgspp paqqnqyvhi s s spqntgrt asppaipvhl hphqtmipht Itlgppsqvv mqyadsgshf vpreatkkae s s rlqqaiqa kevlngemek s rrygaps sa dlglgkaggk svphpyes rh vvvhpspsdy s s rdpsgvra svmvlpnsnt paadlevqqa threaspstl ndksglhlgk pghrsyalsp htviqtthsa seplpvglpa taf yagtqpp vigylsgqqq aityagslpq hlvipgtqpl lipvgstdme asgaapaivt s spqfaavph tfvttalpks enfnpealvt qaaypamvqa qihlpvvqsv aspaaapptl ppyfmkgsii qlangelkkv edlktedf iq saeisndlki ds stveried shspgvaviq f avgehraqv svevlveypf fvfgqgws s c cpertsql fd Ipcs klsvgd vcisltlknl kngsvkkgqp vdpasvllkh s kadglags r hryaeqengi nqgsaqmlse ngelkfpekm glpaapf Itk ieps kpaatr krrwsapes r kleksedepp Itlpkpslip qevkiciegr snvgk (SEQ ID N0:7), a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.

In one embodiment, the gene encodes sodium channel protein type 2 subunit alpha isoform

1 (Homo sapiens) having NCBI Reference Sequence: NP 0010352321 : maqsvlvppg pds f rf f tre slaaieqria eekakrpkqe rkdeddengp kpnsdleagk slpf iygdip pemvsvpled Idpyyinkkt f ivlnkgkai srf satpaly iltpfnpirk laikilvhsl fnmlimctil tncvfmtmsn ppdwtknvey tf tgiytf es likilargf c ledf tf Irdp wnwldf tvit f ayvtefvdl gnvsalrtf r vlralktisv ipglktivga liqsvkklsd vmiltvf cis vfaliglqlf mgnlrnkclq wppdnssfei nitsf fnnsl dgngttfnrt vsifnwdeyi edkshf yf le gqndallcgn ssdagqcpeg yicvkagrnp nygyts fdtf swaflslf rl mtqdfwenly qltlraagkt ymif fvlvif Igs f ylinli lavvamayee qnqatleeae qkeaef qqml eqlkkqqeea qaaaaaasae srdf sgaggi gvf sesssva sklsskseke Iknrrkkkkq keqsgeeekn drvrksesed sirrkgf rf s legsrltyek rf ssphqsll sirgslf spr rnsraslfsf rgrakdigse ndfaddehst fedndsrrds Ifvphrhger rhsnvsqasr asrvlpilpm ngkmhsavdc ngvvslvggp stltsagqll pegtttetei rkrrsssyhv smdlledpts rqramsiasi Itntmeelee srqkcppcwy kf anmcliwd cckpwlkvkh Ivnlvvmdpf vdlaiticiv Intlfmameh ypmteqf ssv Isvgnlvf tg iftaemflki iamdpyyyf q egwnifdgfi vslslmelgl anveglsvlr s f rllrvf kl akswptlnml ikiignsvga Ignltlvlai ivfifavvgm qlfgksykec vckisndcel prwhmhdf fh s f livf rvlc gewietmwdc mevagqtmcl tvfmmvmvig nlvvlnlfla lllssfssdn laatdddnem nnlqiavgrm qkgidfvkrk iref iqkafv rkqkaldeik pledlnnkkd scisnhttie igkdlnylkd gngttsgigs svekyvvdes dyms f innps Itvtvpiavg esdf enlnte ef ssesdmee skeklnatss segstvdiga paegeqpeve peeslepeac f tedcvrkf k ccqisieegk gklwwnlrkt cykivehnwf etf ivfmill ssgalafedi yieqrktikt mleyadkvf t yifilemllk wvaygf qvyf tnawcwldf 1 ivdvslvslt analgyselg aikslrtlra Irplralsrf egmrvvvnal Igaipsimnv llvclifwli f simgvnlfa gkf yhcinyt tgemfdvsvv nnyseckali esnqtarwkn vkvnfdnvgl gylsllqvat f kgwmdimya avdsrnvelq pkyednlymy lyfvifiifg sf ftlnlfig viidnfnqqk kkfggqdifm teeqkkyyna mkklgskkpq kpiprpankf qgmvfdfvtk qvfdisimil iclnmvtmmv etddqsqemt nilywinlvf ivlftgecvl klislryyyf tigwnifdfv vvilsivgmf laeliekyf v sptlf rvirl arigrilrli kgakgirtll f almmslpal fniglllflv mfiyaifgms nf ayvkrevg iddmfnf etf gnsmiclfqi ttsagwdgll apilnsgppd cdpdkdhpgs svkgdcgnps vgif f fvsyi iis f Ivvvnm yiavilenf s vateesaepl seddf emf ye vwekfdpdat qf ief aklsd f adaldppll iakpnkvqli amdlpmvsgd rihcldilf a f tkrvlgesg emdalriqme erfmasnpsk vsyepitttl krkqeevsai iiqrayrryl Ikqkvkkvss iykkdkgkec dgtpikedtl idklnenstp ektdmtpstt sppsydsvtk pekekf ekdk sekedkgkdi reskk

(SEQ ID NO:8), a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.

In one embodiment, the gene encodes helicase sen ataxin isoform 1 (Homo sapiens) having NCBI Reference Sequence: NP, 055861.3: mstccwctpg gastidf Ikr yasntpsgef qtadedlcyc lecvaeyhka rdelpf lhev Iweletlrli nhf eksmkae igdddelyiv dnngemplfd itgqdf enkl rvplleilky pylllhervn elcvealcrm eqancs f qvf dkhpgiylfl vhpnemvrrw ailtarnlgk vdrddyydlq evllclf kvi elgllespdi ytssvlekgk lillpshmyd ttnyksywlg icmlltilee qamdslllgs dkqndfmqsi Ihtmereadd dsvdpfwpal hcfmvildrl gskvwgqlmd pivaf qtiin nasynreirh irnssvrtkl epesylddmv tcsqivynyn pektkkdsgw rtaicpdycp nmyeemetla svlqsdigqd mrvhnstf Iw f ipfvqslmd Ikdlgvayia qvvnhlysev kevlnqtdav cdkvtef f 11 ilvsvielhr nkkclhllwv ssqqwveavv kcaklpttaf trssekssgn cskgtamiss Islhsmpsns vqlayvqlir sllkegyqlg qqslckrfwd klnlflrgnl slgwqltsqe thelqsclkq iirnikf kap pcntfvdlts ackispasyn keeseqmgkt srkdmhclea ssptf skepm kvqdsvlika dntiegdnne qnyikdvkle dhllagsclk qssknifter aedqikistr kqksvkeis s ytpkdctsrn gpergcdrgi ivstrlltds stdalekvst snedf slkdd alaktskrkt kvqkdeicak Ishvikkqhr kstlvdntin Idenltvsni esfysrkdtg vqkgdgf ihn Isldpsgvld dkngeqksqn nvlpkekqlk neelvifsfh ennckiqefh vdgkelipf t emtnasekks spf kdlmtvp esrdeemsns tsviysnltr eqapdispks dtltdsqidr dlhklsllaq asvitfpsds pqnssqlqrk vkedkrcf ta nqnnvgdtsr gqviiisdsd dddderilsl ekltkqdkic lerehpeqhv stvnskeekn pvkeektetl f qf eesdsqc fefesssevf svwqdhpddn nsvqdgekkc lapianttng qgctdyvsev vkkgaegiee htrprsisve ef ceievkkp krkrsekpma edpvrpsssv rnegqsdtnk rdlvgndf ks idrrtstpns riqrattvsq kkssklctct epirkvpvsk tpkkthsdak kgqnrssnyl scrttpaivp pkkf rqcpep tstaeklglk kgprkayels qrsldyvaql rdhgktvgvv dtrkktklis pqnlsvrnnk klltsqelqm qrqirpksqk nrrrlsdces tdvkragsht aqnsdifvpe sdrsdynctg gtevlansnr kqlikcmpse petikakhgs patddacpln qcdsvvlngt vptnevivst sedplgggdp tarhiemaal kegepdsssd aeednlfltq ndpedmdlcs qmendnykli elihgkdtve veedsvsrpq leslsgtkck ykdclettkn qgeycpkhse vkaadedvf r kpglpppask plrpttkif s skstsriagl sksletssal spslknkskg iqsilkvpqp vpliaqkpvg emknscnvlh pqspnnsnrq gckvpf ges k yfpssspvni llssqsvsdt fvkevlkwky emf Inf gqcg ppaslcqsis rpvpvrfhny gdyfnvf fpl mvlntf etva qewlnspnre nf yqlqvrkf padyikywef avyleecela kqlypkendl vf laperine ekkdterndi qdlheyhsgy vhkf rrtsvm rngktecyls iqtqenfpan Inelvncivi sslvttqrkl kamsllgsrn qlaravlnpn pmdf ctkdll tttseriiay Irdfnedqkk aietayamvk hspsvakicl ihgppgtgks ktivgllyrl Itenqrkghs densnakikq nrvlvcapsn aavdelmkki ilef kekckd kknplgncgd inlvrlgpek sinsevlkf s Idsqvnhrmk kelpshvqam hkrkef Idyq Idelsrqral crggreiqrq eldeniskvs kerqelaski kevqgrpqkt qsiiileshi icctlstsgg lllesaf rgq ggvpf scviv deagqsceie tltplihrcn klilvgdpkq Ipptvismka qeygydqsmm arfcrlleen vehnmisrlp ilqltvqyrm hpdiclfpsn yvynrnlktn rqteaircss dwpf qpylvf dvgdgserrd ndsyinvqei klvmeiikli kdkrkdvs f r nigiithyka qktmiqkdld kefdrkgpae vdtvdaf qgr qkdcvivtcv ransiqgsig f laslqrlnv titrakyslf ilghlrtlme nqhwnqliqd aqkrgaiikt cdknyrhdav kilklkpvlq rslthpptia pegsrpqggl psskldsgfa ktsvaaslyh tpsdskeitl tvtskdperp pvhdqlqdpr llkrmgievk ggiflwdpqp sspqhpgatp ptgepgfpvv hqdlshiqqp aavvaalssh kppvrgeppa aspeastcqs kcddpeeelc hrrearaf se geqekcgset hhtrrnsrwd krtleqedss skkrkll (SEQ ID N0:9), a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.

In one embodiment, the gene encodes neurogenic locus notch homolog protein 3 precursor (Homo sapiens) having NCBI Reference Sequence: NP 000426.2: mgpgargrrr rrrpmspppp pppvralpll lllagpgaaa ppcldgspca nggrctqlps reaaclcppg wvgercqled pchsgpcagr gvcqs svvag tarf s crcpr gf rgpdcslp dpcls spcah garcsvgpdg rflcs cppgy qgrs crsdvd ecrvgepcrh ggtclntpgs f rcqcpagyt gplcenpavp capspcrngg tcrqsgdlty dcaclpgf eg qncevnvddc pghrclnggt cvdgvntync qcppewtgqf ctedvdecql qpnachnggt cfntlgghs c vcvngwtges csqniddcat avcfhgatch drvas f ycac pmgktgllch Iddacvsnpc hedaicdtnp vngraictcp pgf tggacdq dvdecsigan pcehlgrcvn tqgs f Icqcg rgytgprcet dvneclsgpc rnqatcldri gqf tcicmag f tgtycevdi decqs spcvn ggvckdrvng f s ctcpsgf s gstcqldvde castpcrnga kcvdqpdgye crcaegf egt Icdrnvddcs pdpchhgrcv dgias fs cac apgytgtrce sqvdecrsqp crhggkcldl vdkylcrcps gttgvncevn iddcasnpct f gvcrdginr ydcvcqpgf t gplcnveine cas spcgegg s cvdgengf r clcppgslpp Iclppshpca hepcshgicy dapggf rcvc epgwsgprcs qslardaces qpcraggtcs sdgmgfhctc ppgvqgrqce llspctpnpc ehggrcesap gqlpvcs cpq gwqgprcqqd vdecagpapc gphgictnla gs fs ctchgg ytgps cdqdi ndcdpnpcln ggs cqdgvgs f s cs clpgfa gprcardvde clsnpcgpgt ctdhvas f tc tcppgyggfh ceqdlpdcsp s s cfnggtcv dgvns fs clc rpgytgahcq headpcls rp clhggvcsaa hpgf rctcle s f tgpqcqtl vdwcs rqpcq nggrcvqtga yclcppgwsg rlcdirslpc reaaaqigvr leqlcqaggq cvdeds shyc vcpegrtgsh ceqevdpcla qpcqhggtcr gymggymcec Ipgyngdnce ddvdecasqp cqhggs cidl varylcs cpp gtlgvlcein eddcgpgppl dsgprclhng tcvdlvggf r ctcppgytgl rceadinecr sgachaahtr dclqdpgggf rclchagf sg prcqtvlspc esqpcqhggq crpspgpggg Itf tchcaqp fwgprcerva rs crelqcpv gvpcqqtprg prcacppgl s gps crs fpgs ppgasnas ca aapclhggs c rpaplapf f r cacaqgwtgp rceapaaape vseeprcpra acqakrgdqr cdrecnspgc gwdggdcsls vgdpwrqcea Iqcwrl fnns rcdpacs spa clydnfdcha ggrertcnpv yekycadhf a dgrcdqgcnt eecgwdgldc asevpallar gvlvltvllp peellrs sad f Iqrlsailr tslrf rldah gqamvfpyhr pspgseprar relapevigs vvmleidnrl clqspendhc fpdaqsaady Igalsaverl dfpyplrdvr gepleppeps vpllpllvag avlllvilvl gvmvarrkre hstlwfpegf slhkdvasgh kgrrepvgqd algmknmakg eslmgevatd wmdtecpeak rlkveepgmg aeeavdcrqw tqhhlvaadi rvapamaltp pqgdadadgm dvnvrgpdgf tplmlas f eg galepmptee deaddtsasi isdlicqgaq Igartdrtge talhlaarya radaakrlld agadtnaqdh sgrtplhtav tadaqgvf qi lirnrstdld armadgstal ilaarlaveg mveeliasha dvnavdelgk salhwaaavn nveatlallk ngankdmqds keetpl flaa regsyeaakl lldhf anrei tdhldrlprd vaqerlhqdi vrlldqpsgp rsppgphglg pllcppgaf 1 pglkaaqsgs kks rrppgka glgpqgprgr gkkltlacpg plads svtl s pvdsldsprp fggppaspgg fplegpyaaa tatavslaql ggpgraglgr qppggcvls l gllnpvavpl dwarlpppap pgps f llpla pgpqllnpgt pvspqerppp ylavpghgee ypaagahs sp pkarf Irvps ehpyltpspe spehwaspsp pslsdwsest pspatatgam atttgalpaq plplsvps sl aqaqtqlgpq pevtpkrqvl a (SEQ ID NO: 10), a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.

In one embodiment, the gene encodes eurogenic locus notch homolog protein 3 precursor

(Homo sapiens) having NCBI Reference Sequence: NP 000426.2: mgpgargrrr rrrpmspppp pppvralpll lllagpgaaa ppcldgspca nggrctqlps reaaclcppg wvgercqled pchsgpcagr gvcqs svvag tarf s crcpr gf rgpdcslp dpcls spcah garcsvgpdg rflcs cppgy qgrs crsdvd ecrvgepcrh ggtclntpgs f rcqcpagyt gplcenpavp capspcrngg tcrqsgdlty dcaclpgf eg qncevnvddc pghrclnggt cvdgvntync qcppewtgqf ctedvdecql qpnachnggt cfntlgghs c vcvngwtges csqniddcat avcfhgatch drvas f ycac pmgktgllch Iddacvsnpc hedaicdtnp vngraictcp pgf tggacdq dvdecsigan pcehlgrcvn tqgs f Icqcg rgytgprcet dvneclsgpc rnqatcldri gqf tcicmag f tgtycevdi decqs spcvn ggvckdrvng f s ctcpsgf s gstcqldvde castpcrnga kcvdqpdgye crcaegf egt Icdrnvddcs pdpchhgrcv dgias fs cac apgytgtrce sqvdecrsqp crhggkcldl vdkylcrcps gttgvncevn iddcasnpct f gvcrdginr ydcvcqpgf t gplcnveine cas spcgegg s cvdgengf r clcppgslpp Iclppshpca hepcshgicy dapggf rcvc epgwsgprcs qslardaces qpcraggtcs sdgmgfhctc ppgvqgrqce llspctpnpc ehggrcesap gqlpvcs cpq gwqgprcqqd vdecagpapc gphgictnla gs fs ctchgg ytgps cdqdi ndcdpnpcln ggs cqdgvgs f s cs clpgfa gprcardvde clsnpcgpgt ctdhvas f tc tcppgyggfh ceqdlpdcsp s s cfnggtcv dgvns fs clc rpgytgahcq headpcls rp clhggvcsaa hpgf rctcle s f tgpqcqtl vdwcs rqpcq nggrcvqtga yclcppgwsg rlcdirslpc reaaaqigvr leqlcqaggq cvdeds shyc vcpegrtgsh ceqevdpcla qpcqhggtcr gymggymcec Ipgyngdnce ddvdecasqp cqhggs cidl varylcs cpp gtlgvlcein eddcgpgppl dsgprclhng tcvdlvggf r ctcppgytgl rceadinecr sgachaahtr dclqdpgggf rclchagf sg prcqtvlspc esqpcqhggq crpspgpggg Itf tchcaqp fwgprcerva rs crelqcpv gvpcqqtprg prcacppgl s gps crs fpgs ppgasnas ca aapclhggs c rpaplapf f r cacaqgwtgp rceapaaape vseeprcpra acqakrgdqr cdrecnspgc gwdggdcsls vgdpwrqcea Iqcwrl fnns rcdpacs spa clydnfdcha ggrertcnpv yekycadhf a dgrcdqgcnt eecgwdgldc asevpallar gvlvltvllp peellrs sad f Iqrlsailr tslrf rldah gqamvfpyhr pspgseprar relapevigs vvmleidnrl clqspendhc fpdaqsaady Igalsaverl dfpyplrdvr gepleppeps vpllpllvag avlllvilvl gvmvarrkre hstlwfpegf slhkdvasgh kgrrepvgqd algmknmakg eslmgevatd wmdtecpeak rlkveepgmg aeeavdcrqw tqhhlvaadi rvapamaltp pqgdadadgm dvnvrgpdgf tplmlas f eg galepmptee deaddtsasi isdlicqgaq Igartdrtge talhlaarya radaakrlld agadtnaqdh sgrtplhtav tadaqgvf qi lirnrstdld armadgstal ilaarlaveg mveeliasha dvnavdelgk salhwaaavn nveatlallk ngankdmqds keetpl flaa regsyeaakl lldhf anrei tdhldrlprd vaqerlhqdi vrlldqpsgp rsppgphglg pllcppgaf 1 pglkaaqsgs kks rrppgka glgpqgprgr gkkltlacpg plads svtl s pvdsldsprp fggppaspgg fplegpyaaa tatavslaql ggpgraglgr qppggcvls l gllnpvavpl dwarlpppap pgps f llpla pgpqllnpgt pvspqerppp ylavpghgee ypaagahs sp pkarf Irvps ehpyltpspe spehwaspsp pslsdwsest pspatatgam atttgalpaq plplsvps sl aqaqtqlgpq pevtpkrqvl a (SEQ ID NO: 11), a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.

Accordingly, variants of the sequences disclosed above are envisioned. For example, a variant may include one or more conservative amino acid substitutions-that is, for example, aspartic-glutamic as acidic amino acids; lysine/arginine/histidine as polar basic amino acids; leucine/isoleucine/methionine/valine/alanine/proline/glycine non-polar or hydrophobic amino acids; serine/threonine as polar or hydrophilic amino acids. Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide- containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting peptide, polypeptide or fusion polypeptide. Whether an amino acid change results in a functional peptide, polypeptide or fusion polypeptide can readily be determined by assaying the specific activity of the peptide, polypeptide or fusion polypeptide.

Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic; trp, tyr, phe. The disclosure also envisions a peptide, polypeptide or fusion polypeptide with nonconservative substitutions. Non-conservative substitutions entail exchanging a member of one of the classes described above for another.

Exemplary Ligand and CD46 Binding Sequences

In one embodiment, the adenoviral fiber/knob binds to hCD46 (membrane cofactor protein isoform 1 precursor (Homo sapiens) NCBI Reference Sequence: NP .002380.3, see below, ^mePPP^{rre c}P fpswrfpgll laamvlllys f sdaceeppt f eameligkp kpyyeigerv dykckkgyf y ipplathtic drnhtwlpvs ddacyretcp yirdplngqa vpangtyef g yqmhf icneg yyligeeily celkgsvaiw sgkppicekv Ictpppkikn gkhtf sevev f eyldavtys cdpapgpdpf sligestiyc gdnsvws raa peckvvkcrf pvvengkqi s gf gkkf yyka tvmf ecdkgf yldgsdtivc dsnstwdppv pkclkvlpps stkppalshs vsts sttksp as sasgprpt ykppvsnypg ypkpeegild sldvwviavi viaivvgvav icvvpyrylq rrkkkgtylt dethrevkf t si

(SEQ ID NO:4), a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.

In one embodiment, the adenoviral fiber/knob comprises

MTKRVRLSDSFNPVYPYEDESTSQHPFINPGFI SPNGFTQSPDGVLTLNCLTPLTTTGGPLQLKVGGGLIVDDTDGT LQENIRATAPITKNNHSVELSIGNGLETQNNKLCAKLGNGLKFNNGDICIKDSINTLWTGIKPPPNCQIVENTDTND GKLTLVLVKNGGLVNGYVSLVGVSDTVNQMFTQKSATIQLRLYFDSSGNLLTDESNLKI PLKNKSSTATSEAATSSK AFMPSTTAYPFNTTTRDSENYIHGICYYMTSYDRSLVPLNI SIMLNSRTI SSNVAYAIQFEWNLNAKESPESNIATL TTSPFFFSYIREDDN

(SEQ ID NO:5), a different isoform of the protein, or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto In one embodiment, the adenoviral fiber/knob comprises:

MTKRVRLSDSFNPVYPYEDESTSQHPFYNPGFI SPNGFTQSPDG

VLTLKCLTPLTTTGGSLQLKVGGGLTVDDTDGTLQENIRATAPITKNNHSVELSIGNG

LETQNNKLCAKLGNGLKFNNGDICIKDSINTLWTGINPPPNCQIVENTNTNDGKLTLV

LVKNGGLVNGYVSLVGVSDTVNQMFTQKTANIQLRLYFDSSGNLLTEESDLKI PLKNK

SSTATSETVASSKAFMPSTTAYPFNTTTRDSENYIHGICYYMTSYDRSLFPLNI SIML

NSRMI SSNVAYAIQFEWNLNASESPESNIATLTTSPFFFSYITEDDN

(SEQ ID NO:6), or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.

In one embodiment, the adenoviral fiber/knob comprises:

MTKRVRLSDSFNPVYPYEDESTSQHPFINPGFI SPNGFTQSPDG VLTLNCLTPLTTTGGPLQLKVGGGLIVDDTDGTLQENIRVTAPITKNNHSVELSIGNG

LETQNNKLCAKLGNGLKFNNGDICIKDSINTLWTGIKPPPNCQIVENTDTNDGKLTLV

LVKNGGLVNGYVSLVGVSDTVNQMFTQKSATIQLRLYFDSSGNLLTDESNLKI PLKNK

SSTATSEAATSSKAFMPSTTAYPFNTTTRDSENYIHGICYYMTSYDRSLVPLNI SIML

NSRTI SSNVAYAIQFEWNLNAKESPESNIATLTTSPFFFSYIREDDN

(SEQ ID NO: 12), or a polypeptide having at least 80%, 82%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 97%, 98% or 99% amino acid sequence identity thereto.

Thus, variants of the sequences disclosed above are envisioned. For example, a variant may include one or more conservative amino acid substitutions-that is, for example, aspartic- glutamic as acidic amino acids; lysine/arginine/histidine as polar basic amino acids; leucine/isoleucine/methionine/valine/alanine/proline/glycine non-polar or hydrophobic amino acids; serine/threonine as polar or hydrophilic amino acids.

Conservative amino acid substitution also includes groupings based on side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. For example, it is reasonable to expect that replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the properties of the resulting peptide, polypeptide or fusion polypeptide. Whether an amino acid change results in a functional peptide, polypeptide or fusion polypeptide can readily be determined by assaying the specific activity of the peptide, polypeptide or fusion polypeptide.

Amino acid substitutions falling within the scope of the invention, are, in general, accomplished by selecting substitutions that do not differ significantly in their effect on maintaining (a) the structure of the peptide backbone in the area of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu;

(4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic; trp, tyr, phe.

The disclosure also envisions a peptide, polypeptide or fusion polypeptide with nonconservative substitutions. Non-conservative substitutions entail exchanging a member of one of the classes described above for another.

Subjects

The subject may be any animal, including a human and non-human animal. Non-human animals include all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, cows, horses, chickens, amphibians, and reptiles, although mammals are envisioned as subjects, such as non-human primates, sheep, dogs, cats, cows and horses. The subject may also be livestock such as, cattle, swine, sheep, poultry, and horses, or pets, such as dogs and cats.

In one embodiment, subjects include human subjects suffering from or at risk for the medical diseases and disorders described herein. The subject is generally diagnosed with the condition by skilled artisans, such as a medical practitioner.

The methods described herein can be employed for subjects of any species, gender, age, ethnic population, or genotype. Accordingly, the term subject includes males and females, and it includes elderly, elderly-to-adult transition age subjects, adults, adult-to-pre-adult transition age subjects, and pre-adults, including adolescents, children, and infants.

Examples of human ethnic populations include Caucasians, Asians, Hispanics, Africans, African Americans, Native Americans, Semites, and Pacific Islanders. The methods may be more appropriate for some ethnic populations such as Caucasians, especially northern European populations, as well as Asian populations.

The term subject also includes subjects of any genotype or phenotype as long as they are in need of treatment, as described above. In addition, the subject can have the genotype or phenotype for any hair color, eye color, skin color or any combination thereof. The term subject includes a subject of any body height, body weight, or any organ or body part size or shape.

The invention will be described by the following non-limiting examples.

Examples

Example 1

Introduction

Due to the clinical successes of viral vector-mediated gene therapy (1,2), it is an attractive strategy to treat many CNS disorders. Although adeno-associated virus vectors (AAV) have many positives, which include the most clinical successes for FDA approved gene therapies (2,3), major drawbacks of both AAV and lentiviral vectors (LVV) are the packaging capacity of ~5 kb and ~9 kb respectively (1,4,5), rendering them incompatible as approaches to treat the root cause of many genetic CNS disorders that are due to mutations in genes that encode large cDNAs (6,7, 8,9). Therefore, AAV approaches are not viable strategies as the cDNAs simply do not fit within AAV. Furthermore, LVV’s relative size to its foreign DNA payload will result in significantly low viral titers (10,11), which severely limits their utility. Although Herpes- Simplex Virus Type 1 (HSV- 1) amplicon vectors have a large packaging capacity, these vectors suffer from serious safety concerns and are not viable options for use in the clinic (12).

Adenoviral vectors have tremendous potential for treatment of CNS disorders and currently being used in human clinical trials (13,14). The most commonly used Ad vectors used in the CNS are based on Ad Group C Serotype 5 (Ad5), which transduces cells via Coxsackie- adenovirus receptor (CAR) (15). However, these Ad5 vectors are unable to transduce many neuronal cell types that are dysfunctional in many CNS disorders. In particular, many hereditary cerebellar disorders are due to Purkinje cell (PC) dysfunction or degeneration (7,16,17). Since PCs mediate the only information outflow from cerebellar cortex and due to PC-related pathologies observed in cerebellar disorders, they are key cell-type to target to treat these disorders (18). However, CAR expression in the mouse and human cerebellum is very low (19,20) and an extremely small subset of human PCs express CAR (21). Furthermore, in vivo injections of Ad5 resulted in little to no transduction of PCs (22,23), while transduction of mouse dissociated cerebellar cultures showed that only 1.6% of PCs were transduced with an Ad5 vector (24). As a result, for ~25 years (22-25) the use of Ad vectors in the cerebellum has been abandoned. Therefore, a novel 1st generation Ad that can transduce PCs and other cerebellar cell types is needed before Ad gene therapy approaches can be developed to treat cerebellar disorders.

The development of chimeric Ad5 vectors, which contain fibers from other Ad serotypes is well-established in generating Ad vectors that transduce refractory cell-types26,27. Group B Ads use the human CD46 receptor (hCD46)28, which has ubiquitous expression in human cells (29,30). Since the Ad capsid proteins are the key determinants of viral vector tropism, 1^st generation Ad5/Group B fiber chimeric vectors were generated from Serotypes 21, 35, or 50, designated as Ad5/21, Ad5/35, and Ad5/50, respectively. Using a validated preclinical humanized mouse model expressing hCD46 (hCD46tg) (31) in conjunction with stereotactic delivery to the cerebellum, it was found that the three chimeric vectors transduced PCs in a hCD46-dependent manner. It was also observed that Ad5/35 and Ad5/50 injection into the deep cerebellar nuclei (DCN) resulted in widespread retrograde transduction of PCs and that a PC-specific promoter led to increased PC transduction (32). Additionally, it was determined that both vectors transduced Bergmann glia, granule cells, and cerebellar afferent mossy fibers independently of hCD46. Taken together, the establishment of these chimeric Ad vectors serves as platform technology for development of high-capacity helper dependent Adenoviral (HdAd) vectors as HdAd production relies on the use of the capsid proteins of a 1st generation helper virus Ad vector. Since HdAd has a large ~36 kb packaging capacity, HdAd vectors with hCD46 dependent tropism are useful for viral vector gene therapy approaches to treat, for example, hereditary cerebellar ataxias and other neurological disorders, including as those requiring expression of large transgenes or multiple transgene cassettes.

Materials and Methods

Animals

Human CD46 transgenic mice (hCD46tg also known as MYII) on C57/B16J background were purchased from the Jackson Laboratory, Bar Harbor, ME (B6.FVB-Tg(CD46)2Gsv/J, RRID:IMSR_JAX:004971) and housed at the University of Iowa Office of Animal Resources facilities under standard laboratory conditions. These mice carry the YAC-CD46 transgene containing full-length human CD46 gene expressed under its endogenous promoter (31). The mice were bred under a scheme to produce offspring that are either wild type or heterozygous for the transgene. Littermates aged from P30 to P60 of both genotypes and sexes were used for the study. The genotyping was done to the tissue obtained from earmark clippings using a standard PCR protocol described by the Jackson Laboratory. All animal procedures were approved by the University of Iowa Institutional Animal Care and Use Committee (Protocol # 0122358). Viral Vectors

El, E3 -deleted 1st generation adenoviral vectors were created under the 2-plasmid rescue or the RAP Ad systems according to standard protocols (63,64). Transgene expression cassettes were constructed in shuttle plasmids via restriction cloning and consisted of either CMV or L7- 6(32) promoters in combination with the fluorescent reporter proteins mClover3, or mCherry, or mScarlet and chimeric intron, and simian virus polyadenylation signal (SV40polyA) sequences. Genomic plasmids for Group B fiber chimeras were constructed from adenovirus Serotype 5 genomic plasmids by excising the shaft and knob sequences of the fiber domain and replacing those with shaft and knob sequences of Serotype 21 (Acc. no.: AY601633.1, bp:31541-32377), or 387 Serotype 35 (Acc. no.: AY128640.2, bp:30961-31797), or Serotype 50 (Acc. no.: AY737798.1, bp:31544-32380). This was achieved by digesting the Ad5 genomic plasmids with Cas9 nuclease (NEB, Ipswich, MA) targeted by sgRNAs 389 spanning the fiber domain with sequences 5’ -gggactctcttgaaacccat-3 ’ (SEQ ID NO: 16) upstream and 5’ -cttaggtgttatattccaca-3 ’ (SEQ ID NO: 17) downstream. The sgRNAs were synthesized using the NEB EnGen sgRNA Synthesis Kit according to the manufacturer’s protocol and purified in spin columns (Monarch - NEB, Ipswich, MA). Upon completion, the Cas9 reactions were used as vector DNA in In-Fusion cloning (TakaraBio, San Jose, CA) reactions to insert synthetically manufactured DNA fragments (IDT, Coralville, IA) consisting of fiber domains of Ad21, or Ad35, or Ad50 and terminal 15 bp homology arms. The location and the sequence integrity of the inserts were confirmed by Sanger sequencing. Following co-transfection of genomic and shuttle DNA, all vectors, including the Ad5/GroupB chimeras were serially amplified successfully in the 1st generation Ad5 producer cell line HEK293. The vectors were then purified based on density by sequential centrifugation in cesium chloride according to standard protocols (63).

The identities of purified vectors have been confirmed via restriction digestion of PCR fragments amplified using a combination of common primers spanning the fiber domains (upstream: 5’ -ttgtatcccccaatgggtttcaag (SEQ ID NO: 18), downstream: 5’- gacaggaaaccgtgtggaatataac (SEQ ID NO: 19)). The viral DNA from purified stocks of each vector was extracted in 0.1% SDS at 56 °C for 10 mins, spin column purified (NucleoSpin - Macher eyNagel, Duren, Germany), and PCR amplified via standard Taq polymerase reaction (EconoTaq - Lucigen, Middleton, WI). PCR products were then digested by the restriction enzyme AfUII (NEB, Ipswich, MA) and visualized on 1% agarose gel stained with ethidium bromide. The extracted viral DNA was also used to determine the physical viral titers via absorbance measurement at 260 nm under a UV-Vis spectrophotometer (NanoDrop One - ThermoFisher, Waltham, MA). The titer (viral particles (VP) / ml) was calculated according to standard methods (63). The infectious titers (transducing units (TU) / ml) were determined using a droplet digital PCR system (QX200 ddPCR System - Bio-412 Rad, Hercules, CA) by end-point amplification of viral DNA extracted from infected HeLa cells via TaqMan assay (ThermoFisher, 414 Waltham, MA). The infectious titers of the vectors were as follows; Ad5 CMV mCherry: 6xlO¹⁰ TU/ml, Ad5/21 CMV mClover3: IxlO¹¹ TU/ml, Ad5/35 CMV mClover3: 6xlO¹⁰ TU/ml, Ad5/50 CMV mClover3: IxlO¹¹ TU/ml, Ad5/50 CMV mScarlet: IxlO¹¹ TU/ml, Ad5/50 L7-6 mScarlet: 3xlO¹⁰ TU/ml.

Stereotactic Injections

Mice were anesthetized using a gas anesthesia system (Rothacher, Heitenried, Switzerland) with 5% isoflurane/Ch mixture and placed on a stereotactic frame (Kopf, Tujunga, CA). Anesthesia was maintained throughout the surgery between 1.5-2.5%. Adequate depth of anesthesia was confirmed by the lack of pedal withdrawal to a toe pinch. Following the scalp application of the local anesthetic (0.5% lidocaine / 0.25% bupivacaine mixture - 5 mg/kg), the skull was exposed via an anterior/posterior incision and cerebellum was accessed through craniectomy of a 0.7 mm burr hole. A glass capillary needle coupled with a nanoliter injector (Neurostar, Tubingen, Germany) and loaded with viral vector solution was lowered to coordinates relative to lambda A/P:-2.00 mm, M/L:+1.80 mm, D/V:+2.00 mm for lobular injections targeting the right simple lobule and to A/P:-2.00 mm, M/L:+1.80 mm, D/V:+3.40 mm for DCN injections targeting the right interposed nucleus under guidance of a robot stereotactic system (Neurostar, Tubingen, Germany). 1 pl viral vector solution containing 109 VP from each co-mixed vector was infused into the right cerebellar hemisphere at a rate of 100 nl/min and the needle was slowly withdrawn at a speed of 1 mm/min. For the quantification of transduction efficiencies, co-mixed vector titers were matched at 2xl0⁷ TU each vector. The incision was closed with veterinary tissue adhesive (Med-Vet, Mettawa, IL) and mice were administered Meloxicam SR (2 mg/kg) for postoperative pain management. Following surgical procedures, mice were housed in an ABSL2c facility and daily monitored for adequate recovery for 5 days.

Tissue Processing

Mice were anesthetized with isoflurane and euthanized by decapitation. The brain tissue was removed, and right hemispheres were immediately dissected. For the BaseScope assay, dissected tissue was drop fixed in 10% neutral buffered formalin (NBF) at RT and processed into paraffin blocks via standard embedding protocols. 5 pm sagittal sections were cut using a microtome (HM325 - ThermoFisher, Waltham, MA), mounted on silanized glass slides, and stored at 4 °C until further processing. For immunofluorescence protocols, injected right hemispheres were drop fixed in 4% paraformaldehyde (PF A) at 4 °C, cerebellum tissue was further dissected, and serially sectioned into series of 12 using a vibratome (VT1200S - Leica, Nussloch, Germany). 50 pm sagittal cut free-floating sections were preserved at 4 °C in phosphate-buffered saline (PBS) containing 0.02% sodium azide until further processing. RNA in situ Hybridization: BaseScope Assay and Brightfield Microscopy

5 pm sections were deparaffinized by series of washes in xylene and hydrated in decreasing concentrations of ethanol washes according to standard protocols. BaseScope assay (ACDBio, Newark, CA) was performed following the standard protocol published by the manufacturer using a custom 1ZZ probe designed to target nucleotides 360-410 of human CD46 (Acc. No.: NM_172351.3) and not to cross detect endogenous mouse transcripts. Chromogenically developed sections (fast red) were counterstained with hematoxylin (VectorLabs, Newark, CA) and coverslipped with Cytoseal XYL (Epredia, Kalamazoo, MI). Tiled RGB photomicrographs were captured from whole mount sections under lOx magnification via brightfield microscopy (DM6 - Leica, Wetzlar, Germany).

Immunofluorescence and Confocal Imaging

50 pm free-floating sections were treated overnight with 0.01 M sodium citrate (pH=6.0) at 60 °C for antigen retrieval and then permeabilized for 1 hour in 0.5% Triton-X/PBS. Sections were blocked in 10% normal goat serum (Jackson ImmunoResearch, West Grove, PA) or with 5% BSA (RPI, Mount Prospect, IL) and incubated in primary antibodies overnight at 4 °C. Following biotinylated secondary antibody (VectorLabs, Newark, CA) incubation for 2 hours at RT, antigen antibody complexes were fluorescently labeled with DyLight649 tagged streptavidin (VectorLabs, Newark, CA) during a 1-hour incubation at RT. For dual immunolabeling experiments, antigen primary antibody complexes were visualized with anti-rabbit Alexa 488 and anti-goat Alexa 647 secondary antibodies (Jackson ImmunoResearch, West Grove, PA). Sections were counterstained with DAPI and coverslipped with Aqua-Mount (Epredia, Kalamazoo, MI). The primary antibodies used were rabbit anti-mouse-Pcp2 C-term. (5 pg/ml, AP6356b - Abcepta, San Diego, CA), goat anti-human-CD46 (0.5 pg/ml, AF2005 -R&D Systems, Minneapolis, MN), rabbit anti-NeuN (1.5 pg/ml, 26975-1-AP - Proteintech, Rosemont, IL), rabbit anti-slOOp (2.5 pg/ml, 15146- 1-AP - Proteintech, Rosemont, IL). For the vGlutl immunofluorescence, a primary antibody directly conjugated to Alexa647 was used with the secondary antibody omitted (single domain anti-vGlutl-Alexa647, 1 :500, N1602-AF647-L - NanoTag, Gottingen, Germany). Coverslipped sections were imaged using a confocal laser scanning microscope (LSM880 - Zeiss, Jena, Germany) under laser lines 405 nm for DAPI, 488 nm for mClover3 vectors and Alexa488, 561 nm for mCherry and mScarlet vectors, and 633 nm for DyLight649 and Alexa647 immunolabeled molecular markers. For Purkinje cell analyses, photomicrographs were acquired under a 20x objective, whereas a 40x objective was used for granule cell, mossy fiber, and Bergmann glia images.

Transduced Purkinje Cell Number Analysis Fluorescent photomicrographs of serially sectioned hCD46tg cerebellum acquired as tiled whole sections with 2 pm z-stacks were analyzed with the open-source imaging package Fiji. The transduced Purkinje cells (PCs) were quantified from all sections within a sagittal series of 12, corresponding to an unbiased representation of the right cerebellar hemisphere from its lateral to medial margin. A researcher, blind to the injected viral vectors visually determined labeled PCs from partial images of 350 pm by 350 pm generated from tiled whole section scans by a custom cell counter script developed for Fiji. Through the researcher’s input, the script recorded the number of transduced PCs by each vector and the number of PCs, on which multiple vector signal co-localizes. The total number of PCs transduced in a section series for each mouse was used for statistical analysis. Statistical Analysis

All statistical analyses were performed using Prism statistical package (GraphPad, Boston, MA). Data were checked for distribution via Shapiro-Wilk normality test. To compare normally distributed group data, unpaired Student’s t-test with Welch’s correction for unequal standard deviations was performed. A p-value of less than 0.05 was considered statistically significant for all analyses and indicated with p>0.05 was represented with ‘ns’. Data are summarized as 497 mean (SD).

Results

Purkinje Cells in the hCD46tg Cerebellum Express hCD46. hCD46 transgenic mice (hCD46tg) are considered an excellent preclinical model for assessing Ad vectors that utilize hCD46 to transduce cells (33-35). Although human PCs express hCD46 (36,37) and measles virus infects similar human CNS cell populations, which include the cerebellum in the hCD46tg mouse (31), it was unknown if PCs in hCD46tg mice express hCD46. Therefore, to determine hCD46 expression in PCs, BaseScope and immunofluorescence (IF) were performed on cerebellar sections from the hCD46tg mice. The results from the BaseScope assay demonstrated that hCD46 mRNA is expressed throughout the hCD46tg brain and in PCs, while wild-type mice did not express hCD46 (Fig. 4A/B). Subsequently, IF was performed against hCD46 together with L7/Pcp2, a PC-specific molecular marker, to validate hCD46 expression at the protein level in the hCD46tg mouse cerebellum. It was found that hCD46 expression by PCs and other cell-types in the molecular and granular cell layers of the hCD46tg cerebellum (Fig. 4D). Antibody specificity was confirmed via lack of signal in cerebellar sections of wild-type mice (Fig. 4C). Since the immunofluorescence confirmed the BaseScope results, hCD46tg mice were used to investigate hCD46-dependent Ad vector transduction in the cerebellum.

Generation of Group B Chimeric Adenoviral Vectors. The Adenovirus fiber, which consists of the shaft and knob region, is a key determinant of vector tropism (26,38). Group B Ads use hCD46 as their primary attachment receptor (28). Since Ad5 chimeras, which contain fibers from other Ad serotypes, can transduce cells refractory to Ad5, and can be produced to high titers (26,27), 1st generation El, E3-deleted Ad5/Group B fiber “chimeric vectors” were generated. To do so, the Ad5 fiber was replaced with the Group B Serotype 50, or 35, or 21 fiber (Fig. 4G). Ad50 and Ad21 are in the same fiber knob clade with protein sequences differing by a single amino acid, while Ad35 is in a distinct fiber knob clade. In addition, Ad21 and A50 are Group Bl, while Ad35 is a Group B2 virus. Furthermore Ad5/35 chimeric vectors efficiently transduce hCD46tg mouse hematopoietic stem cells (33-35). After successful cloning of Ad5/21, Ad5/35, Ad5/50 genomic plasmids, these chimeric vectors were produced to high titers using standard protocols for 1st generation Ad production (See Materials and Methods). Afterwards, the chimeric vector identities were confirmed with restriction digestion of PCR fragments of fiber regions amplified from isolated viral DNA from purified stocks (Fig. 4E,F).

Ad5/21, Ad5/35, Ad5/50 Group B Chimeras Transduce Purkinje Cells.

To investigate if the chimeric vectors transduced PCs, stereotactic injections into the cerebellar cortex of the hCD46tg mouse and wild-type controls were performed. To do so, Ad5/50, or Ad5/35, or Ad5/21 (CMV mClover3) were individually co-mixed with equal viral particles of Ad5 (CMV mCherry) and were stereotactically injected into the PC somatic cell layer in the cerebellar simple lobule of adult mice. Viral particle numbers were used since these were qualitative experiments to determine if the chimeric vectors could transduce Purkinje cells or not. Subsequently, 7 days later, the animals were sacrificed, cerebellar section were generated, and immunofluorescent labeling was used against the PC-specific marker, L7/Pcp2, to analyze colocalization of mClover3 and the PCs (Fig. 5A). It was found that Ad5 transduction (mCherry signal) in the cerebellar cortex did not overlap with L7/Pcp2 in wild-type or in hCD46tg tissue (Fig. 5B). In contrast, mClover3 was detected within cells that were positive for L7/Pcp2 in hCD46tg cerebellar tissue from mice injected with either Ad5/21 (Fig. 5C, right column), or Ad5/35 (Fig. 5D, right column), or Ad5/50 (Fig. 5E, right column). However, we did not find any overlap of mClover3 and L7/Pcp2 signal in the wild-type mice with Ad5/21 (Fig. 5C, left column), or Ad5/35 (Fig. 5D, left column), or Ad5/50 (Fig. 5E, left column). Based on these results, it was concluded that Ad5- derived Group B chimeric vectors can transduce PCs in the hCD46tg mouse and transduction is hCD46-dependent.

Injections of Ad5/35 and Ad/50 in the Deep Cerebellar Nuclei result in Purkinje Cell transduction. Ad5 vectors are capable of transducing neurons through retrograde axonal transport (22,39-42). Since the deep cerebellar nuclei (DCN) are the convergence point of all PC axonal terminals (7), the ability to transduce PCs through retrograde transport has the potential to induce widespread PC transduction as an alternative to multiple injections into many cerebellar lobules. Ad5 vectors can transduce PCs at extremely low levels through injection into the DCN (22,23). Therefore, to investigate if our chimeric vectors were capable of high levels of retrograde transduction of PCs, these vectors were injected into the DCN of hCD46tg mice (Fig. 6A) using a similar approach to the cerebellar lobule injections. For these experiments, Ad5/35 and Ad5/50 were the focus, since Ad50 and Ad21are in the same fiber knob clade with protein sequences differing by a single amino acid, while Ad35 is in a distinct fiber knob clade. In addition, Ad21 and A50 are Group Bl, while Ad35 is a Group B2 virus. Absence of mCherry signal in the cerebellar cortex indicated that Ad5 was unable to transduce PCs via retrograde transduction (Fig. 6B). However, it was found that mClover3 expression co-localized with L7/Pcp2 signal in cerebellar sections of animals injected with either Ad5/35 (Fig. 6C) or Ad5/50 (Fig. 6D). Based on the lack of Ad5 transduction in the cerebellar cortex and the absence of transduction by the chimeric vectors in cell types other than PCs, it was concluded that Ad5/35 and Ad5/50 are capable of transducing PCs through retrograde axonal transport.

Since wild-type Ad50 may have higher affinity for hCD46 than wild-type Ad35 it was determined if potential differences in affinities for the hCD46 receptor between Ad5/35 and Ad5/50 impacted retrograde transduction efficiency. This was done by creating an Ad5/50 CMV mScarlet chimeric vector and co-mixing equal transducing units (TU) of both Ad5/50 CMV mScarlet and Ad5/35 CMV mClover3 vectors and injecting into the DCN of hCD46tg mice. Equal TUs of each chimeric vector were used since we wanted to directly compare the Ad5/35 and Ad5/50 efficiencies to transduce Purkinje cells. Then the number of PCs transduced by each vector were quantified from serially cut cerebellar sections (Fig. 7A). It was found that there were no significant differences in the total number of PCs transduced between the two vectors (Fig. 7B). However, it was found that only 28.84% (1.579%) of PCs were positive for both mClover3 and mScarlet fluorescence (Fig. 7C). Furthermore, DCN injections of both vectors resulted in widespread PC transduction from the most lateral to the most medial sagittal serial section (Fig. 7D). Therefore, the data indicate that a single DCN injection was sufficient to transduce PCs distant to the injection site. Finally, it was concluded that neither vector is superior in transducing PCs through DCN injection in the hCD46tg tissue.

The Purkinje Cell Specific Promoter Improves Purkinje Cell Transduction Efficiency. Promoters are factors that define transduction properties as they impact transgene expression specificity and expression levels. While the CMV promoter leads to ubiquitous high- level expression from viral vectors, use of cell-type specific promoters has been demonstrated to lead to increased transduction of specific cell-types (43,44). The L7-6 promoter which is a derivative of the L7 promoter is a minimal promoter with high specificity to PCs in the cerebellum (32). Therefore, to determine if one could increase PC transduction with the L7-6 promoter, an Ad5/50 L7-6 mScarlet vector was created. Subsequently, its transduction efficiency was compared to Ad5/50 CMV mClover3 via DCN co-injections that delivered equal TU of each vector. Since the L7-6 promoter is weaker than the CMV promoter, the mice were sacrificed 28-days after injection to ensure gene expression from both promoters (Fig. 8 A). By quantifying the number of PCs transduced by each vector from serial cerebellar sections, it was found that Ad5/50 L7-6 mScarlet led to significantly higher number of PCs transduced compared to Ad5/50 CMV mClover3 (820.3 (73.10) vs 559.7 (24.17), p=0.0173, t-test) (Fig. 8B). Although, both vectors transduced PCs in the cerebellar cortex, it was observed that only 22.36% (5.821%) of PCs were co-transduced by the two vectors (Fig. 8C). Further analysis revealed that the transduction patterns of the two vectors differed significantly from each other. At the DCN injection site, numerous cell bodies were labeled by Ad5/50 CMV vector, while we rarely observed any signal from the Ad5/50 L7-6 vector that is consistent in size and shape with transduced cell somata (Fig. 8D). Based on this data, it was concluded that L7-6 promoter results in higher PC transduction efficiency and as reported previously (32), restricts the transgene expression to PCs.

Ad5/35, Ad5/50: hCD46-Independent Tropism in The Cerebellar Cortex.

Despite their ability to bind hCD46, Group B Ads can utilize a variety of other cell surface receptors for transduction (45). The results from lobular injections in the wild-type mice showed that Group B chimeras did not transduce PCs (Fig. 5C-E), however we observed viral transduction in other cell-types. Therefore, we characterized transduction of Ad5/35 CMV mClover3 and Ad5/50 CMV mClover3 using immunofluorescence against cell-type specific molecular markers. These vectors were co-injected with Ad5 CMV mCherry with equal viral particles for each vector into the cerebellar simple lobule as these were qualitative experiments (Fig. 9-11). Previously, it has been reported that Ad5 vectors can efficiently transduce Bergmann glia (24). Therefore, to examine if the Ad5/35 and Ad5/50 vectors transduced Bergmann glia, slOOp was used as a nonspecific glial marker and identified Bergmann glia based on their location and morphology. All vectors including Ad5, transduced Bergmann glia both in the wild-type and hCD46tg mice (Fig. 9A-C). Since Ad5 vectors can transduce granule cells in vitro and in organotypic cultures (24), we next examined granule cell transduction. To do so, we used NeuN as a marker of granule cells in the cerebellar cortex. It was found that Ad5/35 and Ad5/50 vectors transduced low numbers of granule cells in a scattered pattern (Fig. 10B/C) both in the wild-type and in the hCD46tg tissue, but Ad5 signal did not co-localize with NeuN signal (Fig. 10A). Finally, since Ad5 vectors and the Group B chimeras are capable of retrograde transduction, we examined afferent mossy fibers that make glutamatergic synapses in the granule cell layer of multiple cerebellar lobules. We observed cell bodies transduced by the Group B chimeras in various nuclei such as the ECU: external cuneate nucleus, RN: reticular nuclei, PRN: pontine reticular nucleus in the pons and medulla, which are part of several brainstem nuclei, where mossy fibers originate from (46,47) (Fig. 11A left). Consistent with their axonal topology (46,47), we detected mClover3 labelled mossy fibers in the injected simple lobule (Fig. 11A solid frame - middle) and in various noninjected lobules (Fig. 11A dashed frame - right). Since the transduction signal from mossy fiber terminals are partly masked by somatic transduction at the injected lobule, we further examined non-injected lobules using vGlutl immunostaining. Although we did not detect mCherry signal in mossy fiber terminals, which indicated lack of Ad5 transduction (Fig. 1 IB), we observed both Ad5/35 and Ad5/50 driven mClover3 signal co-localized with vGlutl in the wild-type and in the hCD46tg tissue (Fig. 11C/D). Taken together, we conclude that chimeric vectors can transduce various cell types independent of the hCD46 expression and exhibit distinct tropism from Ad5. Discussion

Herein it is demonstrated that Ad5/Group B fiber chimeras, Ad5/50, Ad5/35, and Ad5/21 efficiently transduce Purkinje cells (PC) in vivo in a humanized hCD46 transgenic (hCD46tg) mouse model. In addition, we found Ad5/35 and Ad5/50 were capable of efficient PC transduction through retrograde transport and were able to transduce other cell-types in the cerebellum. Since hCD46 is ubiquitously expressed in human cells, these chimeric viral vectors can be used as platform technology for the development of HdAd gene therapy approaches not only for hereditary cerebellar ataxias but also for other human neurological disorders. The hCD46tg model

The studies relied on the established hCD46tg mouse, which exhibits hCD46 expression patterns and levels that mimics expression patterns observed in humans (31). More importantly, hCD46 expression in this mouse line renders them susceptible to measles infections and infects similar human CNS cell populations, which include the cerebellum (31). The immunofluorescence characterization showed that hCD46tg mouse PCs express hCD46, which is similar to human PCs (036,37). Since every human enucleated cell expresses hCD46 (29,30), the ability of the Ad5/Group B fiber chimeras to transduce PCs in hCD46tg mice indicate that these vectors will have tremendous clinical potential. Prior work has demonstrated that the Ad5/35 and HdAd5/35 vectors successfully transduce hematopoietic stem cell populations from hCD46tg mice and nonhuman primates (33-35,48). Furthermore, intravenous administration of HdAd5/35 vectors in hCD46tg mice and nonhuman primates led to similar biodistribution between the two animals (49). Therefore, this mouse be used in conjunction with other neurological disorder mouse models as preclinical platforms for the development of gene therapy approaches that utilize HdAd5/Group B fiber chimeras or HdAd Group B vectors.

Ad5/21, Ad5/35 and Ad5/50 transduce Purkinje Cells in the hCD46tg mouse model.

The finding that the Ad5/21, Ad5/35, and Ad5/50 transduce PCs in the hCD46tg mice but not in wild-type mice indicates that hCD46 expression is sufficient for PC transduction. However, we did not find differences in the efficiency of different chimeras in transducing PCs. Therefore, the data indicate that that potential differences between Ad21, Ad35, and Ad50 fiber binding affinities to hCD46 are not key determinants in PC transduction. Although hCD46 is expressed on every enucleated cell, Ad21, Ad35, and Ad50 have distinct cell-type tropism (45). Although differences in Group B fiber affinity for hCD46 is proposed to underpin the distinct tropisms (50), there is no consensus if the fiber binding affinity is a key determinant of cell-type transduction. Based on in vitro hCD46 binding assays, the Ad35 fiber is considered to have high affinity to hCD46, while Ad21 fiber displays low affinity (50). In contrast, assays examining the attachment of Group B Ads to hCD46 expressing cell lines showed that the number of Ad50 and Ad21 viruses attached to the hCD46 expressing cells is significantly higher than Ad35 (28). Furthermore, in cell lines Ad viral vectors with engineered Ad35 fibers with higher affinity for hCD46 did not lead to increased transduction efficiencies compared to the native Ad35 fiber (51). However, it is speculated that other steps that contribute to transduction are impacted by the difference in fiber binding affinity leading to higher transduction in certain tissues (51).

Retrograde transduction of Purkinje cells with Ad5/35 and Ad5/50

The cerebellum has multiple lobules, which creates a complicated architecture in which to target maximal transduction coverage of all PCs. Thus, although targeting PCs through multiple lobular injections would be feasible, an alternative delivery approach would be ideal. Since all PCs input into the DCN, the ability to utilize retrograde transport to transduce PCs is an attractive strategy. Ad5 vectors are capable of transducing neurons via retrograde transduction with Canine Adenoviral vectors being one of the most efficient (52). Adenovirus fibers are involved in intracellular trafficking and uncoating kinetics (51), which may influence retrograde transduction efficiency. While Ad5 vectors are capable of retrograde transduction and can transduce PCs through retrograde transduction via DCN injection, it is inefficient (22,23). It was found that injection of Ad5/35 and Ad5/50 into the DCN led to highly efficient transduction of PCs with broader transduction areas compared to the injection into the lobule. Furthermore, the data showed that Ad5 injection into the DCN did not lead to PC transduction in both mouse genotypes, but DCN injection of Ad5/35 and Ad5/50 in the wild-type mice did not lead to PC transduction. Therefore, the data indicates that Ad35 and Ad50 fibers are responsible for retrograde transduction and that PC presynaptic terminals contain hCD46 receptors. Since Ad fibers target Ad virions to distinct trafficking pathways, it is likely that the Ad35 and Ad50 fibers target the chimeric vectors to trafficking pathways that lead to retrograde transduction. Based on previous work, the Ad35 fiber targets Ad5/35 chimeras to the late endosomal and lysosomal compartments and it is postulated that this leads to proximity to nucleus (51,53). Since both Ad5/50 and Ad5/35 led to similar levels of retrograde transduction, this suggests that both Ad50 and the Ad35 fiber target the chimeras to the same trafficking pathways. However, future studies need to be carried out to determine the trafficking of these vectors in PCs and neurons in general. Additionally, it was found that only -29% of all transduced PCs were co-transduced by both Ad5/35 and Ad5/50 via DCN injection. While this may be due to distinct trafficking pathways, it is possible that the competition between the viral vectors for the hCD46 receptor and potential downregulation of hCD46 after binding is the mechanism behind partial co-localization (51,54).

Since the Ad5/35 and Ad5/50 vectors transduced PCs over a broad area via injection into the DCN, this suggests that DCN injection could be a clinically translatable approach for HdAd300 based cerebellar gene therapy. Although intraparenchymal injections are an invasive approach, this is currently utilized in numerous AAV clinical and preclinical trials and other clinical trials for viral vector delivery to the human brain (55-58) as gene therapy approaches for neurological disorders such as Huntington’s disease (NCT04120493, NCT05243017), Parkinson’s disease (NCT01621581, NCT04167540, NCT00195143, NCT00643890, NCT05603312, NCT00229736), multiple system atrophy (NCT04680065), Alzheimer’s disease (NCT05040217), Batten disease (NCT01161576), and Tay-Sachs disease (NCT04669535). Although the DCN is a deep tissue area, MRI-guided delivery has significantly improved precision and minimized tissue damage, successfully delivering AAV vectors to deep brain areas such as the substantia nigra pars compacta and ventral tegmental area (57). Convection-enhanced delivery (CED) of viral vectors through intraparenchymal injection has been optimized to transduce large areas of the brain (59). CED has been safely used to treat aromatic-L-amino acid decarboxylase deficiency (AADC) with an AAV2-AADC gene therapy approach in children (55).

The L7 promoter leads to increased Purkinje cell transduction efficiency.

The study utilized the CMV promoter to evaluate PC transduction efficiency. While the CMV promoter is a strong ubiquitous promoter, promoters that drive strong ubiquitous expression can lead to toxicity due to overexpression of the gene of choice or undergo gene silencing (2,60). Therefore, the use of more endogenous cell promoters is ideal. The L7-6 promoter is the smallest version of the L7 promoter that confers PC specificity, which in the context of AAV and LVV drives high levels of expression in PCs (32). The L7-6 promoter is more restrictive to PCs compared to the smaller L7-4 version of the promoter (32). The data showed that similar to AAV and LVV, the L7-6 promoter leads to an increase in specificity of expression compared to the CMV promoter. Although the L7-6 version leads to high PC specificity, it may not be an ideal promoter to utilize for treating monogenic cerebellar disorders as the mutant genes underlying these disorders are expressed in a wide-variety of cell-types that may also contribute to the disease phenotype. Thus, L7-4 promoter, which leads to expression in multiple cerebellar cell-types in addition to PCs may be ideal to be utilized in the context of HdAd gene therapy approaches. In addition, CaMKIIa or neuron-specific synapsin promoters were found to slightly improve PC transduction efficiencies compared to the ubiquitous CAG promoter (61). However, further investigations are needed to directly compare the L7-6 with L7-4 or CaMKIIa, or synapsin promoters to determine the most suitable promoter for HdAd-based cerebellar gene therapy. Ad5/35 and Ad5/50 transduce multiple cell types in the cerebellum.

While the study was largely focused on the ability of the chimeric vectors to transduce PCs, we found that the chimeric vectors led to transduction of Bergmann glia, mossy fibers, and granular cells. Interestingly, it was found that transduction was not dependent on hCD46 as similar transduction patterns with Ad5/35 and Ad5/50 were seen in both the hCD46tg and wild-type mouse. However, transduction of granule cells and mossy fibers was dependent on the fiber of Ad35 and Ad50 as this transduction pattern was not seen with Ad5. In contrast, Ad5/35, Ad5/50, and Ad5 transduced Bergmann glia. In the absence of fiber-mediated interaction, adenoviruses can utilize direct interaction between the penton base and cell surface integrins for attachment (62). Since all the chimeras utilize the Ad5 penton, this interaction could mediate uptake of the viral particles into the cells. Subsequently, once inside the cell the Ad35 and Ad50 fibers could target the viral particles to a different trafficking pathway than Ad542, which leads to differences in transduction. In support of this hypothesis, it was found that mossy fibers were transduced. Mossy fibers are long range afferent fibers that originate from multiple nuclei outside of the cerebellum synapsing on multiple cerebellar lobules (22,23). Thus, the only way for mossy fibers to be mClover3 positive including in the non-injected lobules would be via retrograde transduction of their cell bodies distant to the injection site. Although the Ad35 and Ad50 fibers are considered to be hCD46 exclusive (45), it is possible that these fibers can interact with an alternative receptor distinct from hCD46 or CAR.

Implication for HdAd gene therapy approaches for cerebellar and neurological disorders.

Taken together, the work has identified that Group B Ad fibers are sufficient to transduce PCs and other cell types in the hCD46tg mouse model. HdAd production relies on a 1st gen Ad helper virus and the capsid proteins of the 1st gen helper virus are the key determinant of viral vector transduction. Therefore, these 1st gen Ad chimeric vectors can be quickly developed into Helper viruses for HdAd production to develop HdAd Group B chimeric vectors. More importantly, the use of Ad5/Group B chimeric vectors opens up new avenues for Ad and HdAd gene therapy applications, not only for cerebellar disorders but also for multiple neurological disorders that cannot be addressed by AAV and LVV.

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All publications, patents and patent applications are incorporated herein by reference. While in the foregoing specification, this invention has been described in relation to certain preferred embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details herein may be varied considerably without departing from the basic principles of the invention.

Claims

WHAT IS CLAIMED IS:

1. A delivery vehicle comprising a molecule that binds to human CD46 (hCD46) comprising: an adenovirus fiber/knob region that binds to hCD46 and optionally comprises one or more prophylactic or therapeutic gene products or nucleic acid encoding one of more of the prophylactic or therapeutic gene products.

2. The delivery vehicle of claim 1, wherein the delivery vehicle is a virus comprising the adenoviral fiber/knob region that binds to hCD46, such as recombinant virus.

3. The delivery vehicle of claim 2, wherein the virus or recombinant virus is a adenovirus comprising a adenoviral genome that encodes the adenoviral fiber/knob region.

4. The delivery vehicle of claim 3 which is El" and/or E3‘ or HdAd

5. The delivery vehicle of claim 3 or 4, wherein the adenovirus genome is from a human adenovirus.

6. The delivery vehicle of any one of claims 1 to 5, wherein at least a portion of the adenovirus fiber/knob region is from a human adenovirus.

7. The delivery vehicle of any one of claims 3 to 6, wherein the recombinant adenovirus is helper dependent.

8. The delivery vehicle of any one of claims 1 to 7, wherein the fiber/knob region is from a Group B adenovirus.

9. The delivery vehicle of any one of claims 1 to 7, wherein the fiber/knob region is from serotype 50, serotype 21 or serotype 35.

10. The delivery vehicle of any one of claims 1 to 9, further comprising a nucleic acid encoding a prophylactic or therapeutic gene product.

11. The delivery vehicle of claim 10, wherein the nucleic acid comprises a CACNA1 or ATM gene.

12. The delivery vehicle of any one of claims 1 to 11 which comprises a liposome.

13. The delivery vehicle of any one of claims 1 to 12 which comprises a nanoparticle or microparticle.

14. The delivery vehicle of any one of claims 1 to 13, wherein the gene product comprises a protein.

15. The delivery vehicle of claim 14 which comprises a liposome, a nanoparticle or microparticle comprising the protein.

16. The delivery vehicle of claim 14 which comprises a cell-type specific promoter, such as a neural cell specific promoter.

17. The delivery vehicle of any one of claims 1 to 14 which encodes a plurality of prophylactic or therapeutic gene products.

18. The delivery vehicle of any one of claims 1 to 2 or 6 to 14 or 17 which is a rAAV or lentivirus vector.

19. A pharmaceutical composition comprising the delivery vehicle of any one of claims 1 to 18 and optionally a carrier.

20. A method to prevent, inhibit or treat a disease or disorder of the central nervous system, comprising administering to a mammal in need thereof a composition comprising an effective amount of the delivery vehicle of any one of claims 1 to 18 or the composition of claim 19.

21. The method of claim 20, wherein the composition is systemically administered.

22. The method of claim 20, wherein the composition is locally administered.

23. The method of claim 20, wherein the composition is administered to the central nervous system.

24. The method of claim 20, wherein the composition is intracranially administered.

25. The method of any one of claims 20 to 24, wherein the mammal is a human.

26. The method of any one of claims 20 to 25, wherein the mammal has an ataxia.

27. The method of any one of claims 20 to 25, wherein the mammal has Huntington’s disease.

28. The method of any one of claims 20 to 25, wherein the mammal has amyotrophic lateral sclerosis.

29. The method of any one of claims 20 to 25, wherein the mammal has a CaV 2.1 channelopathy.

30. The method of any one of claims 20 to 25, wherein the mammal has familial hemiplegic migraine type 1 (FHM1), episodic ataxia type 2, spinocerebellar ataxia type 6 (SCA6), or fragile X-associated tremor/ataxia syndrome (FXTAS).

31. The method of any one of claims 20 to 25, wherein the gene product comprises CACNA1, Huntingtin, ATM, or ATXN2.