WO2000017375A2

WO2000017375A2 - Method to treat haemophilia by in vivo gene therapy with retroviral vectors

Info

Publication number: WO2000017375A2
Application number: PCT/EP1999/007384
Authority: WO
Inventors: Thierry Vandendriessche; Marinee K. L. Chuah
Original assignee: Vlaams Instituut voor Biotechnologie VIB
Current assignee: Vlaams Instituut voor Biotechnologie VIB
Priority date: 1998-09-23
Filing date: 1999-09-21
Publication date: 2000-03-30
Anticipated expiration: 2001-03-23
Also published as: AU6468199A; WO2000017375A3

Abstract

The present invention relates to a gene transfer system preferably pseudotyped retroviral vectors allowing stable expression of biologically active proteins at therapeutic, physiological or supraphysiological levels. The invention relates particularly to a method to treat haemophilia using said vectors.

Description

Method to treat haemophilia by in vivo gene therapy with retroviral vectors

Field of the invention The invention relates to a gene transfer system more in particular to pseudotyped retroviral vectors, allowing stable expression of biologically active proteins at therapeutical, physiological or supraphysiological level, useful for in vivo gene therapy.

The present invention relates particularly to a method to treat haemophilia using pseudotyped retroviral vectors encoding coagulation factors, such as coagulation factor VIII (FVIII) or coagulation factor IX (FIX). Haemophilia is characterized by spontaneous and prolonged bleeding in the joints, muscle and internal organs. It is potentially life-threatening and is often associated with disabling arthropathy resulting from the recurring joint bleeding episodes. Haemophilia A and B are congenital X-chromosome linked coagulation disorders, due to a deficiency in coagulation factor VIII (FVIII) or factor IX (FIX) respectively, which are normally expressed in the liver. Haemophilia A occurs in 1 in 10,000 whereas haemophilia B affects 1 in 30,000 males. FVIII has no intrinsic enzymatic activity and functions as a cofactor to accelerate the activation of factor X by activated FIX in the presence of calcium and phospholipids. Ultimately, the coagulation cascade leads to the localized generation of thrombin and conversion of fibrinogen to insoluble fibrin polymers, which in conjunction with platelet aggregation maintains hemostasis.

Background of the invention Retroviral vector-mediated transduction of primary cells with the gene encoding coagulation FVIII or FIX offers the potential of long-term gene expression in haemophilia A or haemophilia B patients, respectively and hence phenotypic correction of the bleeding disorder. However, previous reports on ex vivo transduction of hematopoietic stem/progenitor cells with FVIII-retroviral vectors followed by reimplantation in normal (Hoeben et al., 1992) or haemophilic mice (Evans and Morgan, 1998) did not yield detectable FVIII protein and the haemophilic phenotype could not be corrected. Similarly, no FVIII could be detected following subcutaneous transplantation of ex vivo transduced fibroblasts into immunodeficient mice (Hoeben et al., 1992). However, transient FVIII expression levels (for 2 weeks) were obtained following intraperitoneal implantation of neo-organs containing retrovirally transduced fibroblasts (Dwarki et al., 1995). The lack of (persistent) FVIII expression in these studies could be ascribed to several factors including promoter inactivation, high cell mortality or post- transcriptional inhibition of FVIII production.

Though FVIII-retroviral vectors have been utilised to transduce various mammalian cell types ex vivo, direct in vivo gene transfer has been limited by the low vector titer and low FVIII expression levels that are 1000-fold lower in comparison to vectors carrying other cDNA's (Hoeben et al., 1990, 1992, 1993, Israel and Kaufman, 1990, Lynch et al., 1993, Chuah et al. 1995). This is at least partly due to the presence of sequences in the FVIII cDNA that repress its expression, resulting in low levels of F //-specific mRNA (Lynch et al., 1993, Chuah et al., 1995). Whereas some sequences inhibit transcriptional elongation (Koeberl et al., 1995), other elements seem to have an inhibitory effect on transcriptional initiation, presumably because of the presence of A/T rich nuclear matrix attachment regions (MAR) within the A2 (Fallaux et al., 1996) region of the FVIII cDNA. Following conservative mutagenesis of the entire 1.2 kb inhibitory region, vector titer and FVIII expression remained relatively low (Chuah et al. 1995). To overcome this limitation, the retroviral vector design was modified and it was shown that inclusion of an intron upstream of the FVIII cDNA led to a significant increase in FVIII expression and retroviral titer (Chuah ef al., 1995, Dwarki et al., 1995, Chuah et al., 1998). Other drawbacks of first-generation FVIII retroviral vectors that preclude direct in vivo gene transfer applications include their sensitivity to human complement and the difficulty to purify and concentrate large amounts of vectors. The pioneering studies of Friedmann and colleagues (Yee et al., 1994) had shown that pseudotyping Moloney murine leukemia virus (MoMLV) particles with vesicular stomatitis virus G glycoprotein (VSV-G) yields vector preparations with broader host range that can be concentrated to high titers by ultracentrifugation. In this earlier work, pseudotyping required transient transfection techniques to express the VSV-G protein, since the constitutive expression of high levels of VSV-G is toxic in most cells. However, this method of virus production limits large scale production and possible clinical applications, since only small amounts of virus can easily be produced. Connelly et al. (1998) have shown that adenoviral vectors encoding human FVIII can correct the haemophilia A phenotype in FVIII-deficient mice and lead to long-term expression of FVIII. However, FVIII expression was not stable and declined possibly due to an immune response towards the adenovirally transduced cells or to chronic hepatotoxic effects inherent to the use of E1/E3 deleted or E7/E3/E2a-deleted adenoviral vectors (Connelly et al., 1998) as the number of transduced hepatocytes in vivo declined. Moreover, the use of these vectors for achieving long-term expression in haemophilia patients is highly questionable and will likely be hampered by a strong anti-adenoviral immune response.

An alternative gene transfer approach consists of using conjugates composed of the FVIII cDNA, adenoviral particles and transferrin as targeting ligand (Zatloukal et al., 1994). By combining receptor-mediated uptake of the FVIII gene via the transferrin receptor with adenovirus-augmented gene delivery, primary mouse fibroblasts were transfected with a B-domain (Toole et al., 1986) deleted FVIII expression plasmid. When the engineered cells were surgically implanted into mouse spleens, low levels of FVIII could be detected for only one day after intrasplenic administration (Zatloukal et al., 1994). Since the use of these conjugates led to transient expression in vitro and in vivo, it is unlikely that this approach will be useful for the treatment of haemophilia A.

The potential of in vivo gene therapy for haemophilia B with retroviral vectors was evaluated by direct infusion of retroviral vectors containing the canine FIX cDNA into the portal vasculature of haemophilia B dogs (Kay et al., 1993) The animals were subjected to partial hepatectomy prior to the in vivo gene transfer to induce hepatocyte proliferation and constitutively expressed low levels of canine FIX (about 0.1 % of normal levels, typically less than 10 ng/ml) for over 9 months. Persistent low level expression of the clotting factor, resulted in 60% reductions of whole blood clotting - and partial thromboplastin times of the treated animals. A further 10-100-fold increase in production would be required to reach a clinically beneficial range. A novel procedure has been developed for mediating direct in vivo gene transfer into hematopoietic cells (Nelson et al., 1997). The procedure involves injection of irradiated retroviral producer cells, that produce retroviral vectors containing the human FIX cDNA, into the femoral bone marrow cavity in rabbits without preconditioning. The emergence of vector-marked cells in multiple peripheral blood hematopoietic lineages was detected 1 week post- injection and persisted until the animals were sacrificed up to 20 months later. Vector-marked cells were also detected in different hematopoietic tissues including bone marrow, spleen, thymus, and lymph node. Expression of retrovirus-specific messenger RNA by reverse transcription polymerase chain reaction was detected at multiple time points up to 20 months. The FIX protein was expressed in granulocytes, isolated 14 months after the procedure indicating that hematopoietic stem or early progenitor cells had been transduced in vivo. However, the FIX protein could not be detected in the plasma.

WO 98/00542 discloses a non-invasive in vivo gene therapy for haemophilia A using high-titer retroviral vectors. These FVIII retroviral vectors contain an intron upstream of the B-domain (Toole et al., 1986) deleted FVIII cDNA. Using these vectors it was shown that moderate levels of human FVIII can be expressed long-term in juvenile rabbits and in juvenile dogs following direct intravenous injection of high-titer retroviral vectors. However the normal physiological level of FVIII necessary in humans (200 ng/ml) was not reached and since the animals were not haemophilic, it was not possible to unequivocally demonstrate that this approach could be successful to cure haemophilia A. Moreover, when haemophilic dogs were used, the therapeutic effect obtained as shown by a shortening of whole blood clotting time (WBCT) was not maintained in a stable way, since, after the initial effect, the WBCT lengthened and only transient peaks of FVIII activity could be detected corresponding to a maximum of only picogram levels of functional FVIII. These levels are clearly below therapeutic levels in a haemophilia A patient since patients with functional FVIII plasma levels lower or equal than 2 ng/ml FVIII (< 1 %) still suffer from severe haemophilia. In addition anti-human FVIII antibodies were generated in dogs that led to the formation of circulating immune complexes consisting of human FVIII and canine anti-human FVIII antibodies. Hence, though moderate levels of FVIII could be achieved in non- haemophilic animal models like rabbits and dogs these levels of functional FVIII could not be obtained by the method described in WO 98/00542 in haemophilia A dogs since FVIII levels were not therapeutic in these animals (< 2 ng/ml). From the data of WO 98/00542, where nanogram levels of FVIII can be obtained in normal dogs, whereas only picogram levels are obtained in haemophilic dogs, it is not obvious to achieve therapeutic levels in haemophilic animals or haemophilia A patients.

No gene therapy method, in particular methods based on the use of retroviral vectors have shown stable, long-term therapeutic levels of FVIII or FIX and phenotypic correction of the bleeding disorder in clinically relevant haemophilia A or B animal models such as haemophilia A or B mice or dogs. Furthermore, correction of a genetic disease by direct in vivo retroviral injection has not been demonstrated either.

Summary of the invention The present invention provides a method to treat haemophilia A and/or B, using a gene transfer system or vector, more in particular a pseudotyped retroviral vector, to obtain a physiological level of FVIII and/or FIX over a long period of time. This is the first time that stable, long-term therapeutic levels of FVIII or FIX and phenotypic correction of the bleeding disorder by gene therapy in a clinically relevant haemophilia A or B animal model such as haemophilia A or B mice have been demonstrated. Furthermore, it is also for the first time demonstrated that a genetic disease can surprisingly be corrected by direct in vivo retroviral treatment such as retroviral injection. Hereto an intron-based Moloney murine leukemia virus retroviral vector comprising a B-domain (Toole et al., 1986) deleted human FVIII cDNA (designated as MFG-FVIIIDB) was pseudotyped with vesicular stomatitis virus G glycoprotein (VSV-G). The invention is not limited to this specific pseudotyping, but other proteins such as the amphotropic 4070A amphotropic envelope, the Gibbon ape leukemia virus envelope (Miller et al., 1991 ) , the 10A1 envelope (Miller and Chen, 1996), the Sendai virus glycoprotein F (Spiegel et al., 1998), or the RD114 feline endogenous virus envelope (Cosset et al., 1995) may also be used for pseudotyping. Then the pseudotyped vector is concentrated by centrifugation. Other types of concentration, such as ultracentrifugation (Yee et al., 1994, Miyanohara et al., 1995, Sekhar et al., 1996) precipitation (Sekhar et al., 1996), filtration (Kotani et al., 1994, Sekhar et al., 1996), lyophilization (Kotani et al., 1994) and/or chromatography may also be used to concentrate the vector. This high titer vector preparation is then injected, optionally with one or more transduction additives, into new-born recipients. Alternatively recipients may be used in which the target tissue to be transduced is proliferating or is induced to proliferate by factors such as keratinocyte growth factor (Bosch et al., 1996) or hepatocyte growth factor (Kosai et al., 1998). Retroviral vectors derived from retroviruses (see RNA Tumor Viruses, 2^nd Edition, Cold Spring Harbour Laboratory, 1985) including for example onco-retroviruses (e.g. Moloney murine leukemia virus) as well as spumaviruses (e.g. human foamy virus, Russell and Miller, 1996) and lentiviruses (Naldini et al., 1996) such as Human Immunodeficiency Virus, Feline Immunodeficiency Virus (Poeschla et al., 1998), Simian Immunodeficiency Virus or Equine Infectious Anemia Virus, which do not require cell division for stable transduction, can also be used for gene therapy of haemophilia A or B, or for other genetic diseases. Direct injection of high-titer retroviral vectors may also be useful for delivering proteins deficient in other diseases, for example in familial hypercholesterolemia, lysosomal storage disorders (Gaucher's disease) and other hepatic diseases or diseases resulting from plasma protein deficiencies.

Detailed description of the invention Thus the invention concerns a gene transfer system capable to sustain a stable therapeutic level of a biologically active protein such as FVIII, said level is obtained in an animal model or in a human patient suffering from a disease caused by an insufficient level and/or insufficient activity of said protein. To the invention also relates a gene transfer system capable to sustain by in vivo gene therapy a stable therapeutic level of a biologically active protein such as FVIII, said level is obtained in an animal model or in a human patient suffering from a disease caused by an insufficient level and/or insufficient activity of said protein.

On the other hand the invention also concerns a gene transfer system capable to sustain by in vitro gene therapy a stable therapeutic level of a biologically active protein such as FVIII, said level is obtained in an animal model or in a human patient suffering from a disease caused by an insufficient level and/or insufficient activity of said protein. Moreover the scope of the current invention concerns a viral vector capable to sustain by in vivo or in vitro gene therapy a stable therapeutic level of a biologically active protein such as FVIII, said level is obtained in an animal model or in a human patient suffering from a disease caused by an insufficient level and/or insufficient activity of said protein. More in particular the invention relates to a retroviral vector capable to sustain by in vivo or in vitro gene therapy a stable therapeutic level of a biologically active protein such as FVIII, said level is obtained in an animal model or in a human patient suffering from a disease caused by an insufficient level and/or insufficient activity of said protein. An important aspect of the invention is a pseudotyped retroviral vector capable to sustain in vivo a therapeutic, physiological or supraphysiological level of a biologically active protein for at least 30 days, preferentially at least

100 days up to at least 440 days or more.

A further aspect of the invention concerns said pseudotyped retroviral vector in which the biologically active protein is derived form the group of coagulation factors.

Another aspect of the invention is a pseudotyped retroviral vector capable to sustain in vivo a theurapeutic human coagulation factor VIII level in blood plasma and/or other bodily fluids .

In addition the invention concerns a pseudotyped retroviral vector capable to sustain in vivo a human coagulation factor VIII level in blood plasma and/or other bodily fluids of at least 100 ng/ml, preferentially at least 130 ng/ml, more preferentially at least 200 ng/ml and this for at least 30 days, preferentially at least 100 days up to at least 440 days or more.

A further aspect of the invention is a pseudotyped retroviral vector, capable to sustain in vivo a coagulation factor IX level in blood plasma and/or other bodily fluids of more than 10 ng/ml, preferentially more than 50 ng/ml (= therapeutical level of FIX) and more preferentially at least 5000 ng/ml (= physiological level of FIX) and this for at least 30 days, preferentially at least

100 days.

Another aspect of this invention is a method to treat haemophilia A and/or B, using said vectors or gene transfer system encoding coagulation factors such as FVIII and/or FIX respectively.

Still another aspect of the invention is the use of said vector(s) or gene transfer system to prevent induction of inhibitory and/or neutralising antibodies directed against said biologically active protein, in particular coagulation FVIII and/or FIX.

To the invention also belongs the use of a gene transfer system or a vector according to the invention for the manufacture of a medicament to treat haemophilia A and/or B. Definitions

It may be helpful to an understanding of the current invention as disclosed herein to set forth definitions of various terms used.

Biologically active protein means any protein, polypeptide or peptide having a biological function. Said protein can be, amongst others, an enzyme, a cytokine, a signalling molecule, a transcription factor, a cofactor or a structural protein and the like.

Biological activity of factor VIII refers to a function or set of functions performed by the polypeptide or fragments thereof in a biological system or in an in vitro system.

Gene transfer system means any method allowing delivery of genetic information or material in cells. Preferably the system comprises a (non) viral vector allowing to deliver in one or more cells a biologically active protein.

In vivo gene therapy means direct injection, or other delivery method known to the people skilled in the art into an animal or a patient of a vector encoding a biologically active protein resulting in gene transfer of the recipient's cells in vivo.

Physiological level of a biologically active protein means the average level of said protein as determined in a healthy individual. The physiological level of

FVIII is defined as 100-200 ng/ml (200 ng/ml =1 U/ml = 100%) and the physiological level of FIX is defined as 5000 ng/ml (5000 ng/ml = 1 U/ml =

100%).

Supraphysiological level of a biologically active protein means a level of said protein that is significantly higher than the physiological level.

Severe haemophilia A or B is characterized by less than one percent functional FVIII or FIX, respectively (as measured with a functional assay, compared to the normal physiological level).

Moderate haemophilia A or B is characterized by functional FVIII or respectively FIX levels ranging from 1 to 5 percent (as measured with a functional assay, compared to the normal physiological level). Mild haemophilia A or B is characterized by functional FVIII or respectively

FIX levels ranging from 6 to 40 percent (as measured with a functional assay, compared to the normal physiological level).

Therapeutic levels of FVIII or FIX means a level that converts severe to moderate haemophilia A or B, respectively; or preferably severe and moderate to mild haemophilia A or B, respectively; or more preferably severe, mild and moderate haemophilia A or B respectively, to normal in humans or in a clinically relevant haemophilia A or B animal model such as haemophilia A or B mice or dogs. Levels below one percent FVIII (< 2ng/ml) or FIX (< 50 ng/ml) in humans or haemophilia A or B mice or dogs are defined as non-therapeutic.

Therapeutic levels for any other disease caused by an insufficient level and/or activity of a biologically active protein means a production of said biologically active protein in animal models or in human patient, suffering from said disease, that leads to a less severe form of said disease or leads to a correction of the disease.

Pseudotyped retroviral vector means a vector that comprises part of the genome of a retrovirus in a viral particle that comprises preferably a modified envelope or an envelope from another type of virus or from another retroviral strain than said retrovirus.

Vector construct, retroviral vector or recombinant vector refers to a nucleic acid construct which carries and is capable of directing the expression of a nucleic acid molecule of interest.

Factor VIII is a non-enzymatic cofactor found in blood in an inactive precursor form. Precursor factor VIII is converted to the active cofactor, factor Villa, through limited proteolysis at specific sites by plasma proteases, notably thrombin and factor IXa.

Neutralising and/or inhibitory antibodies against human co-agulation factor

FVIII means antibodies that specifically inhibit the FVIII activity, as measured by the Bethesda assay and expressed in Bethesda units (BU). 1 BU is defined as the amount of antibody that leads to a 50% reduction in FVIII activity. A plasma with an activity higher than 0.5 BU is considered to contain inhibitory antibodies.

Inhibition of induction of neutralising and/or inhibitory antibodies means that no such antibodies can be detected using an Elisa test (i.e. level <1 ng/ml using Mab18 as control) and/or the Bethesda assay (i.e. activity < 0.5 BU/ml).

Brief description of the figures

Fig. 1. Functional FVIII expression in haemophilia A mice. Neonatal FVIII- deficient littermates were injected with concentrated VSV₂₉₃-FVIII vector. Mean plasma FVIII activities (n=3) of recipient mice with prolonged FVIII expression (mice #1 to #6 in Table I are depicted by A,v ,l, H, , , respectively) and negative control mouse injected with PBS (IZl).The experiment was repeated twice in 2 independent batches of animals with different vector lots. Mice #1 , #4, #5 (A, H, ) were injected with 1.4 x

10⁸ cfu/ml and mice # 2, #3, #6, (♦,#,^) with 0.9 x 10⁸ cfu/ml VSV₂₉₃-FVIII. Tail-clipping was performed 2 months post-injection except for mice #5 and #6 (3 months). Five out of 13 mice injected with VSV₂₉₃-FVIII vector (#9 to #13, in Table I) and all control mice injected with PBS (9 out of 9) did not yield detectable FVIII.

Fig. 2. Reciprocal correlation between FVIII activity and inhibitor titer in transient FVIII expressors. Functional FVIII expression, (#, H ) and inhibitory antibody titer (O, D ) of mouse #7 (circles) and #8 (squares) (see Table I) were shown at different intervals post-injection of VSV₂₉₃-FVIII.

Fig. 3. Modulation of infectivity of VSV₂₉₃-FVIII.Viral vector producer cells were grown in the presence (+) or absence (-) of tetracycline (Tet), to repress or induce VSV-G expression, respectively, and incubated with (+) or without (- ) neutralizing VSV-G specific Mab prior to transduction of COS-7 cells in vitro or injection in FVIII-deficient neonates. FVIII activity was determined in the 24 hr-conditioned medium of the transduced COS-7 cells and the relative transduction efficiencies were determined by FVIII-specific PCR (B) (lanes 2- 5). PCR was also performed on DNA from liver (lane 7), spleen (lane 8) and lungs (lane 9) of FVIII-deficient recipient mice injected with the same inactivated concentrated vector preparations. Positive control derived from FVIII-containing cells (lane 10) and molecular weight (MW) marker corresponding to the Smart Ladder (lanes 1 & 6) (Eurogentec, Belgium) are included and FVIII (1.1 kb)-specific fragments are indicated.

Fig. 4. Analysis of gene transfer efficiency by quantitative PCR in liver, spleen and lungs (A) and in testis, heart, brain, kidney, stomach and intestine (B). The average vector copy number per diploid genomic equivalent was determined by comparison with a serially diluted linear standard (correlation coefficient r²-- 0.98) ranging from 0.5 to 0 (negative control) copies per diploid genomic equivalent. Organs from individual mice were indicated (#1 , #2, #5, #7 from Fig.1 and Table I). Mouse #1 and #2 were not sacrificed but a liver biopsy was taken instead and decreasing amounts of target DNA were used as template: 200 ng (a), 100 ng (b) and 50 ng (c). A constant amount of total DNA (200 ng) was maintained in the standard and in the liver samples from mouse #1 and #2 by adding spleen DNA from a FVIII-deficient mouse. FVIII (1.1 kb) or control b-actin (0.2 kb)-specific fragments are indicated. The MW marker corresponds to the Smart Ladder.

Fig. 5. Analysis of gene transfer efficiency by quantitative PCR in liver, spleen and lungs of non-expressor (#10, #11 , #13, from Table I) and transient expressor mice (#7). Bands corresponding to the amplified FVIII or control 2-acftn-specific fragments are indicated (1.1 kb, 0.2 kb, respectively). The same standard was used as described in the legend of Fig. 4. The MW marker corresponds to the Smart Ladder for the FVIII-PCR and the 1 kb ladder for β-actin.

Fig. 6. Expression analysis of FVIII mRNA by RT-PCR in transduced organs derived from either FVIII-expressor mice (#1 , #2, #5 from FigJ and Table I) or a PBS-injected FVIII-deficient mouse as negative (-) control. RNA samples with (+) or without (-) RT as controls were shown to exclude genomic DNA amplification and FVIII -specific RT-PCR products were indicated (0.7 kb).

Examples

Example 1

Retroviral vector production and titration

The non-pseudotyped MFG-FVIIIDB vector (Dwarki et al., 1995) was produced and titrated as previuosly described (Chuah et al. 1998). To generate the VSV-G pseudotyped MFG-FVIIIDB retroviral vector, subconfluent 293GPG packaging cells were transduced successively with conditioned medium containing the MFG-FVIIIDB vector in the presence of polybrene (8 microgram/ml) and tetracycline (1 microgram/ml ). The 293GPG cell line expresses VSV-G in a conditional tetracycline-regulated manner. In the absence of tetracycline, VSV-G expression was induced leading to syncytia formation and production of VSV-G pseudotyped vector particles, whereas VSV-G expression was repressed by addition of tetracycline, allowing normal cell growth. Individual clones were obtained by limiting dilution. Clones that expressed the highest levels of FVIII as measured with a functional Coatest (Coatest FVIII, Chromogenix, Molndal, Sweden; Chuah et al., 1998) were induced in the absence of tetracycline and screened for viral production by RNA dot blot analysis (Chuah et al., 1998) since the MFG- FVIIIDB vector did not contain a selectable marker. A random-primed vector- specific probe was derived by PCR as described (Chuah et al., 1998) using the MFG-FVIIIDB plasmid as target and primers spanning a 418-bp region within the packaging sequence (5'-GGGCCAGACTGTTACCACTCCC-3' and 5'-GCGCCTAGAGAAGGAGTGAGGG-3'). Additional controls consisted of serially diluted viral vector supematants with known functional titer based on VSV-G pseudotyped vectors containing a neomycine resistance gene (LXSN) (Miller and Rosman, 1989). Functional titer was determined by transduction of NIH-3T3 cells as previously described (Chuah et al., 1995) and expressed in G418-resistant colony forming units per ml (cfu/ml). To exclude the presence of rearranged proviral sequences, producer clones were subjected to Southern blot analysis (Chuah et al., 1995). Following expansion of the highest producer clone in the presence of tetracycline, semiconfluent cells were seeded in a 10-tray cell-factory (Nalge Nunc Inc.). When the plates were 80-90% confluent, VSV-G protein expression was induced by growing the cells in medium without tetracycline. The supernatant was harvested at 24 or 48 hr interval over 2 weeks, frozen immediately on dry ice prior to storage at - 80°C and filtered using a 0.45 μm filter before use. Viral concentration was carried out by centrifugation (Bowles et al., 1996) and titer of concentrated vector preparations and yield was determined by RNA dot blot analysis as described above.

The titer of the VSV-G pseudotyped MFG-FVIIIDB vector (designated as VSV₂₉₃-FVIII) was equivalent to 1.8 + 0.7 x10⁶ cfu/ml (mean + SEM, n=7) and was significantly increased (30-fold, p<0.001) compared to the amphotropic MFG-FVIIIDB vector produced by Y-CRIP cells (6 + 3x10⁴ cfu/ml, n=23 ) (Dwarki et al., 1995; Chuah et al., 1998). This increase is most likely due to the stronger CMV promoter used to drive gag and pol expression in the 293GPG cells compared to the LTR promoter in the Y-CRIP packaging cell line. In addition, the VSV₂₉₃-FVIII vector could be concentrated 1000-fold further by centrifugation yielding final average titers equivalent to 1.1 + 0.4 x10⁹ cfu/ml with yields of 63 + 9 % (n=7). The titer achieved represents a significant 10⁶-fold increase in viral titer compared to non-pseudotyped, non- concentrated first-generation FVIII retroviral vectors (Chuah et al., 1995; Hoeben et al., 1990; Israel and Kaufman, 1990). Additional advantages of using these pseudotypes for haemophilia A gene therapy include their increased resistance to human complement inactivation and a reduced likelihood to generate replication competent retroviruses (RCR) (Ory et al., 1996). To assess phenotypic correction, mice were constrained and a 1-cm section of the tail was clipped without subsequent cauterisation. Survival was monitored over time. Example 2 Animal studies

FVIII-deficient mice containing a disruption of the murine FVIII gene in exon- 17 were backcrossed with C57BI/6 mice over 5 generations. Genotyping and phenotypic characterisation of the offspring was performed as described (Bi et al., 1995) and confirmed that all FVIII-deficient mice used contained the disrupted murine FVIII gene. Two to three days old homozygous female and hemizygous male FVIII-deficient littermates (14 mice in total) were injected intravenously over 2 consecutive days with concentrated VSV₂₉₃-FVIII vector at a total dose equivalent to 0.9 or 1.4x10⁸ cfu. Similarly, nine FVIII-deficient littermates were injected with PBS as control. Following injection, topical thrombin was applied to arrest bleeding. One neonate died following injection, due to bleeding at the injection site that could not be arrested by topical thrombin application, but no acute toxicity of the high-titer vector was observed. Plasma samples were obtained from each mouse by retro-orbital bleeding at different intervals for functional human FVIII determination.

Example 3

Analysis of FVIII expression

Biologically active human FVIII was quantified in citrated plasma samples in triplicate from mice by measuring the FVIII-dependent generation of factor Xa from factor X using a chromogenic assay (Coatest FVIII, Chromogenix,

Molndal, Sweden) as previously described (Chuah et al., 1995). Plasma from

FVIII-deficient mice was spiked with human plasma derived FVIII

(Octapharma, Langenfeld, Germany) of known activity as standard to exclude plasma interference. One unit corresponded to 200 ng FVIII/ml (100%) and the sensitivity of the assay was approximately 30 mU/ml. Physiologic FVIII levels were defined as 100-200 ng/ml. Example 4

Detection of anti-FVIII antibodies

Human FVIII-specific antibodies were detected by ELISA as previously described (Connelly et al., 1998) with some modifications. Plates were coated with 3 U/ml human plasma-derived FVIII (Octapharma, Langenfeld, Germany). Serially diluted monoclonal mouse anti-human FVIII antibody (Mab 18) specific for the light chain was used as a control (Gilles et al., 1993). The lowest concentration of this antibody that could still be detected was 0.5 ng/ml. The human FVIII inhibitory antibody titer in the serum samples that scored positive in ELISA was determined with a Bethesda assay as described (Kasper et al., 1975). Inhibition of FVIII activity by serially diluted mouse plasma was measured using a functional FVIII Coatest (Chuah et al., 1995, 1998). One Bethesda Unit (BU) was defined as the amount of antibody that leads to a 50% reduction in FVIII activity.

Example 5

In vivo FVIII expression and phenotype correction

Long-term (>5 months), high-level (>200 mU FVIII/ml) functional human FVIII expression could be detected in 6 of the 13 (46%) animals, 4 of which (31%) had physiologic or supraphysiologic levels (500-12500 mU/ml) (Fig. 1). Human FVIII-specific inhibitory antibodies were not detected by ELISA in these long-term expressors (Table 1 ). Five of the six (83%) high expressors survived an otherwise lethal tail-clipping demonstrating phenotypic correction (Table 1 ) and expressed FVIII for at least 5 months. Some mice were followed-up for > 14 months and still expressed stable, high levels of FVIII. The functional FVIII activity in plasma was completely and specifically inhibited with polyclonal anti-FVIII antibodies from a haemophilia patient and with a monoclonal anti-human FVIII antibody (Mab18) (Gilles et al., 1993). Expression of FVIII was transient in 2 of the 13 (15%) and non-detectable in 5 of the 13 (38%) animals receiving the high-titer vector (Fig. 1 , Table 1). Six of the seven animals with transient or no expression of human FVIII developed FVIII inhibitors as measured by ELISA and Bethesda assays (7 to 350 BU/ml), continued to bleed following tail-clipping and died within a few hours after injury (Table 1 ). Functional human FVIII or human FVIII inhibitory antibodies could not be detected in any of the FVIII-deficient control animals injected with PBS (Table 1) which all died following tail-clipping whereas control C57BI/6 mice survived the tail-clipping.

Persistent, high levels of FVIII expression correlates strongly with the absence of inhibitory antibodies whereas short-term or no FVIII expression was associated with induction of inhibitory antibodies. These observations suggest that high levels of FVIII expression may be required to establish immune tolerance to FVIII in the FVIII-deficient mice. This explanation is consistent with the induction of tolerance to FVIII by repeatedly injecting FVIII proteins at high concentrations in haemophilia A patients (Kreuz et al., 1995) and indicates that induction of inhibitory antibodies to FVIII following gene therapy can be prevented by sufficiently high FVIII expression levels (preferentially physiological or supraphysiological). PCR (polymerase chain reaction) indicated that liver, spleen and lungs were predominately transduced following intravenous injection of high-titer VSV₂₉₃-FVIII, whereas no PCR signal could be detected in ovary, testis, brain, kidney, heart, intestine or thyroid tissue.

Thus neonatal intravenous injection of high-titer FVIII retroviral vectors could lead to efficient secretion of the FVIII protein into the circulation and long-term phenotypic correction of haemophilia A in FVIII-deficient mice. Both the use of VSV-G pseudotyped high-titer vectors and of neonatal recipients in which target tissues contained actively dividing cells (Miyanohara et al., 1995) may have contributed to the observed effects. Human FVIII expression ranged between 20 and 1250% of normal human FVIII levels for at least 14 months post-transduction (Fig. 1) in recipients that did not develop inhibitory antibodies. Example 6

Polymerase chain reaction

Genomic DNA was extracted from different organs by phenol-chloroform extraction. To determine relative transduction efficiencies in the various organs, PCR was performed on 200 ng of DNA using primers specific for the FVIII cDNA spanning the B-domain deletion (5'-GATGAGAACCGAAGCTGG- 3' and 5'-GTCAAACTCATCTTTAGTGGGTGC-3') and β-actin specific primers. PCR was performed using AmpliTaq Gold (Perkin Elmer) by denaturation for 10 min at 95°C, followed by 30 cycles for FVIII (and 28 cycles for β-actin ) of 1 min at 95°C, 1 min at 59°C, 2 min at 72°C and a final extension for 5 min at 72°C, yielding a 1.1 kb FVIII (B-domain deleted)- specific PCR product and a 0.2 kb β-actin specific product. Serially diluted DNA obtained from a producer clone containing 5 integrated FV7//-proviral copies was used as a standard to calculate the average vector copy number per diploid genomic equivalent in the various organs. A constant amount of DNA (200 ng) was maintained in the standard by adding spleen DNA from a FVIII-deficient mouse. Amplified products were separated by gel electrophoresis on 1.5% agarose gels. The intensities of the PCR-amplified vector-specific FV7//-fragment relative to the standard were quantified with a Stratagene Eagle Eye II and NIH Image 1.61/ppc software after background subtraction and β-actin normalization.

To determine which organ expressed FVIII, total RNA was purified using Trizol followed by reverse transcriptase PCR (RT-PCR). Potentially contaminating residual genomic DNA was first eliminated using DNAse I (Life Technologies). The same PCR conditions were used as above except for a different primer pair that specifically primed in FVIII exon 23 (C1 region) (5'- TCTTCTTTGGCAATGTGGATTCAT-3') and the retroviral vector backbone (5'-GTTGAGTCAAAACTAGAGCCTGGACC-3'). Amplified RT-PCR products were also separated by gel electrophoresis on 1.5% agarose gels. Example 7

Inhibition of gene transfer

Viral vector producer cells were grown in the presence or absence of tetracycline, to repress or induce VSV-G expression, respectively. The conditioned medium was concentrated 1000-fold by centrifugation and incubated at 4°C for at least 30 min with or without 10% (v/v) anti-VSV-G Mab (11 , 1-2 mg/ml ascites) (Lyles et al., 1982) prior to transducing COS-7 cells (10⁵ cells/ml) or injection into FVIII-deficient neonates. In vitro transductions were performed by centrifuging vector and target cells at 2600 rpm for 1 hr (32°C) with polybrene (8 mg/ml). Cells were incubated at 32°C and washed the next day. Conditioned medium was collected to determine FVIII activity using a FVIII COAtest. DNA was extracted from the transduced COS-7 cells and from FVIII-deficient recipient mice injected with the inactivated vector preparations and subjected to FVIII-specific PCR to determine relative gene transfer efficiencies.

Table 1. Phenotypic correction of murine hemophilia A. Human FVIII levels (^*) expressed in mU/ml were determined at the time of tail-clipping and anti- FVIII antibodies in the plasma were determined by ELISA as described in the Methods section. The amount of inhibitors in the plasma samples that scored positive for the presence of antibodies by ELISA was determined by Bethesda assays and expressed in Bethesda Units per ml (^$). Undetectable FVIII activity or anti-FVIII antibodies are indicated by (-). Survival of the mice was determined by a tail-clipping assay ^(§). All experimental animals (n=13) and 6 of the 9 control animals injected with PBS were subjected to the tail-clipping assay. Animals that survived were indicated by (+) whereas mice that died within several hours are indicated by (-). The mean values + SEM are shown.

Table 1 PHENOTYPIC CORRECTION OF MURINE HEMOPHILIA A

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Claims

1. Gene transfer system capable to sustain a stable therapeutic level of a biologically active protein such as FVIII, said level is obtained in an animal model or in a human patient suffering from a disease caused by an insufficient level and/or insufficient activity of said protein.

2. Gene transfer system capable to sustain by in vivo gene therapy a stable therapeutic level of a biologically active protein such as FVIII, said level is obtained in an animal model or in a human patient suffering from a disease caused by an insufficient level and/or insufficient activity of said protein.

3. Gene transfer system capable to sustain by in vitro gene therapy a stable therapeutic level of a biologically active protein such as FVIII, said level is obtained in an animal model or in a human patient suffering from a disease caused by an insufficient level and/or insufficient activity of said protein.

4. Viral vector capable to sustain by in vivo or in vitro gene therapy a stable therapeutic level of a biologically active protein such as FVIII, said level is obtained in an animal model or in a human patient suffering from a disease caused by an insufficient level and/or insufficient activity of said protein.

5. Retroviral vector capable to sustain by in vivo or in vitro gene therapy a stable therapeutic level of a biologically active protein such as FVIII, said level is obtained in an animal model or in a human patient suffering from a disease caused by an insufficient level and/or insufficient activity of said protein.

6. Pseudotyped retroviral vector capable to sustain by in vivo gene therapy a therapeutic level of a biologically active protein for at least 30 days, preferentially at least 100 days, said level is obtained in an animal model or in a human patient suffering from a disease caused by an insufficient level and/or insufficient activity of said biologically active protein.

7. Pseudotyped retroviral vector capable to sustain by in vivo gene therapy a physiological or a supraphysiological level of a biologically active protein for at least 30 days, preferentially at least 100 days.

8. Pseudotyped retroviral vector according to claim 6 or 7 in which the biologically active protein belongs to the group of coagulation factors.

9. Pseudotyped retroviral vector capable to sustain in haemophilia A animal models or in haemophilia A patients by in vivo gene therapy a therapeutic FVIII level in the blood plasma and/or other bodily fluid of at least 2 ng/ml, preferably at least 12 ng/ml, more preferably more than 80 ng/ml and most preferably physiological (100-200 ng/ml) or supraphysiological levels (>200 ng/ml).

10. Pseudotyped retroviral vector capable to sustain by in vivo gene therapy a FVIII level in the blood plasma and/or other bodily fluid of at least 130 ng/ml, preferentially at least 200 ng/ml.

11. Pseudotyped retroviral vector according to claim 9 or 10 in which the level is sustained during a period of at least 30 days, preferentially at least 100 days.

12. Pseudotyped retroviral vector according to claim 6-11 in which the retroviral vector is an onco-retroviral vector, preferentially a Moloney murine leukemia virus vector, a spumaviral vector or a lentiviral vector.

13. Pseudotyped retroviral vector according to claim 6-12 wherein the vector is pseudotyped with the vesicular stomatitis virus G-protein, the amphotropic 4070A amphotropic envelope, the Gibbon ape leukemia virus envelope, the 10A1 envelope, the Sendai virus glycoprotein F or the RD114 feline endogenous virus envelope.

14. Pseudotyped retroviral vector according to claim 8 in which the biologically active protein is coagulation factor IX.

15. Method to treat haemophilia A and/or B using a gene transfer system or vector according to claim 1-14.

16. Method to treat haemophilia A and/or B according to claim 15 wherein the vector is injected in a blood vessel of the patient.

17. Method to treat haemophilia A and/or B comprising injecting a high-titer retroviral vector preparation, optionally together with one or more transduction additives, into recipients wherein said retroviral vector is a pseudotyped retroviral vector according to claim 6-14; said retroviral vector is concentrated to high titer by centrifugation and/or ultracentrifugation.

18. Use of a pseudotyped retroviral vector according to claim 6-14 to prevent induction of inhibitory and/or neutralising antibodies directed against a biologically active protein.

19. Use of a pseudotyped retroviral vector according to claim 6-14 to prevent induction of inhibitory and/or neutralising antibodies against coagulation factor FVIII or FIX.

20. Use of a gene transfer system according to claim 1-3 or a vector according to claim 4-14 for the manufacture of a medicament to treat haemophilia A and/or B.