HK1190754A - Personalized production of biologics and method for reprogramming somatic cells - Google Patents

Description

Personalized production of biologicals and methods for reprogramming somatic cells

Cross reference to related applications

The present application claims the priority of us provisional patent application No. 61/429,409, filed on 3/1/2011, and us provisional patent application No. 61/431,376, filed on 10/1/2011. All of the foregoing applications are incorporated herein by reference in their entirety.

Background

There are currently a number of recombinant polypeptides and proteins that are used therapeutically to treat a number of diseases. These recombinant polypeptides and proteins are all commercially produced using primary continuous non-human or human diploid cell lines. For example, some are made using bacteria, such as e.coli (e.coli), while others are made using various ovarian cell lines of yeast or animal origin. Bacteria cannot be used to make certain polypeptides and proteins, especially when glycosylation patterns and other protein modifications are critical to biological receptor binding affinity, biological activity, biodistribution or pharmacokinetics of biologics or immune recognition by the recipient. Chinese hamster ovary cells are one of the most commonly used cell lines currently in biological manufacture when glycosylation is a key variable factor.

Unfortunately, the need for prolonged use of recombinant polypeptides or proteins or for long-term therapy can lead to the production of neutralizing antibodies against the product, thereby rendering the patient less or not responsive to the drug. In some cases, the patient may be taken another drug of the same class, such as a variety of anti-TNF biologicals, such as enbo (Enbrel), also known as Etanercept (Etanercept); remicade (remicade), also known as Infliximab (Infliximab); certolizumab ozogamicin (Certolizumab); and pemetrexed (Humira), also known as D2E7, which are routinely used to treat diseases such as rheumatoid arthritis, juvenile rheumatoid arthritis, psoriasis, psoriatic arthritis, ankylosing spondylitis, ulcerative colitis, and Crohn's disease. However, generating neutralizing antibodies against one particular anti-TNF biologic often predisposes patients to eventually generate neutralizing antibodies against another anti-TNF biologic. In some cases, there is no alternative therapy for the patient, and thus such neutralizing antibody formation would render the patient treatment-less selectable. Even when appropriate treatment options exist, the propensity to generate neutralizing antibodies against other drugs of the same class ultimately means that these patients may not have treatment options.

Other biological products that can be neutralized by human antibodies include: natalizumab (Natalizumab) or Tysabri (Tysabri), an innovative therapy for multiple sclerosis; and Denosumab, a fully human monoclonal antibody directed to nuclear factor kappa-B receptor activator ligand (RANKL), is approved for the treatment of osteoporosis and chemotherapy-induced bone fractures, and may be used for the treatment of breast cancer caused by HRT and hormonal contraceptives. Abatacept (Abatecept) is a CTLA-4 fusion protein approved for rheumatoid arthritis patients who have become refractory to anti-TNF therapy. Although its use is too new to have evidence for the neutralizing antibody induced by abundan, it is also possible that it can induce a human antibody neutralizing antibody response.

Other polypeptides and proteins besides antibody and fusion protein based drugs have also been shown to elicit immune responses in the context of long term therapy. For example, recombinant human erythropoietin elicits the formation of neutralizing antibodies that reduce its efficacy and cause rare hypoplasia syndrome. In addition, the production rate of antibodies to coagulation factor therapy for hemophilia patients is as high as 25% -30% in said patients. This is a general problem in the case of coagulation factors. Recombinant interferon alpha 2a therapy for cancer and hepatitis b is also hampered by the production of neutralizing antibodies to the therapy. The difficulty of long-term growth hormone therapy for small children is also problematic. There have also been reports of the formation of neutralizing antibodies against some insulin products.

Other biopharmaceuticals that have the potential to elicit neutralizing antibodies include whole blood, serum, plasma mixes or other major sources of biological supplies, for example, human albumin, human alpha 1-protease inhibitors, the human antihemophilic Factor/von Willebrand Factor complex, BabyBig (BabyBig) intravenous botulism immunoglobulin, C1 esterase inhibitors, fibrin sealant (fibrin sealant), fibrinogen, intravenous immunoglobulin, subcutaneous immunoglobulin, protein C concentrate, ρ (D) intravenous immunoglobulin, thrombin, von Willebrand Factor/coagulation Factor VIII complex.

Recombinant polypeptides and proteins elicit immune responses and neutralizing antibody production based on a variety of characteristics, including: time frame of biological treatment, time interval of repeated therapy, amino acid composition of biological product and modifications to biological product such as glycosylation, methylation, nitrosylation, sialylation, phosphorylation, sulfation, prenylation, selenization, ubiquitination, vitamin-dependent modifications, protein-binding association, acylation, glycation, three-dimensional conformation, and supercoiling. Thus, there is a need for methods of producing polypeptide protein products with reduced levels of antigenicity in animals treated with biological products.

The teachings of all references cited herein are incorporated herein by reference in their entirety.

Disclosure of Invention

The present invention fills this need by providing methods for producing biological products (e.g., polypeptides or proteins, nucleic acids, viruses, and vaccines) by transfecting or transforming synthetically produced pluripotent stem cells (sppscs) or endogenous pluripotent stem cells (epscs). These cells are derived from the species being treated and are transfected with a vector that expresses the desired biological product and induces expression of the biological product by the transfected or transformed spPSC or ePSC.

The invention further provides a method of producing a recombinant polypeptide or protein, comprising producing a spPSC from an adult cell or isolating an ePSC from an animal, and transfecting or transforming the spPSC or ePSC with a nucleic acid encoding the polypeptide or protein under conditions in which the polypeptide or protein is expressed by the stem cell.

In an alternative embodiment of the invention, cells from the same population as the subject administered the recombinant polypeptide or protein produce sppscs or isolate epscs. Different populations may have different glycosylation patterns and different multiformities from one population to another. The populations are those with the same blood type or tissue type. Thus, according to the invention, recombinant polypeptides and proteins are produced from spPSC or ePSC, wherein the ePSC or spPSC is produced by cells isolated from the same population as the subject to which the polypeptide or protein is administered. The subject will be administered a recombinant polypeptide or protein produced by spPSC or ePSC, wherein the spPSC is produced by a cell from a population belonging to the subject or the ePSC is isolated.

The invention further provides a method of administering a polypeptide or protein to an individual animal, comprising generating a spPSC (such as an Induced Pluripotent Stem Cell (iPSC)) or isolating an ePSC from the cells of the animal and transfecting or transforming the spPSC or ePSC with a nucleic acid encoding the polypeptide or protein under conditions in which the polypeptide or protein is expressed by the pluripotent stem cell, isolating the polypeptide or protein from the induced pluripotent stem cell, and administering the isolated polypeptide or protein to the individual.

The present invention provides a method for personalizing the production of polypeptide or protein therapeutics such that sppscs or epscs are commercially viable and useful. The present invention relates to methods of how to prepare patient-specific sppscs or epscs to make patient-specific polypeptides or proteins to overcome the problem of neutralizing antibody formation that typically occurs in the case of long-term use of polypeptides or proteins. The patient may be any animal, preferably a mammal and more preferably a human. The invention also provides a method for producing a nucleic acid or virus comprising transfecting or transforming a spPSC or ePSC with a vector under conditions to produce a desired nucleic acid or virus.

Furthermore, the present invention provides methods of deriving patient-specific and organ or cell type specific cell lines for the production of closely matched post-translationally modified biologics for therapeutic use. Patient-specific stem cells may be derived using SCNT, induced reprogramming, parthenogenesis, or ANT-OAR reprogramming methods or they may be isolated from the target patient. The pluripotent stem cells thus derived or isolated may be genetically modified to express the therapeutic agent of interest using standard molecular biotechnology procedures, for example using insertion or episomal expression vectors or homologous recombination methods. The genetically modified cell lines can be expanded in culture and banked for periodic bioproduction operations that will be scheduled based on the shelf life of the bioproduct produced (example 2). Alternatively, the derived patient-specific stem cell line may be differentiated in culture to the cell type that normally expresses the highest level of the desired therapeutic protein and then used for biofabrication. Differentiation can be performed for each manufacturing operation or can be performed on a large scale and the differentiated patient-specific cell lines are pooled for subsequent manufacturing operations based on the shelf-life of the therapeutic agent produced (example 3).

In addition, since glycosylation patterns and other post-translational modifications are known to differ between different tissues and cell types, patient-specific stem cell lines can be prepared from adult or somatic cells isolated from organs or cell types endogenously expressing biologics. As an example, SCNT, PGA, ANT-OAR or reprogramming techniques may then be applied to derive pluripotent cell lines for bio-fabrication. The pluripotent stem cells thus derived or isolated can be genetically modified to express a therapeutic agent of interest using standard molecular biotechnology procedures. The genetically modified cell lines can be expanded in culture and banked for periodic bioproduction operations that will be scheduled based on the shelf life of the bioproduct produced (see example 4). Further taking advantage of the 'memory' properties of reprogrammed cells (iPS cells), patient and tissue or cell type specific iPS cells can be induced to differentiate back to their original cell type to more fully form cell lines capable of endogenous post-translational modifications. iPS cells can be genetically modified to express a therapeutic agent of interest either before redifferentiation to the original isolated cell type or after redifferentiation to the original cell type (see example 5).

For example, growth hormone is usually produced in the highest amounts by somatotrophic cells in the anterior pituitary and is additionally highly expressed in cells within the placenta (trophoblast) and tongue, as well as the vulva or anal skin. According to the Human Protein expression profile (Human Protein Atlas), factor VIII Protein expression is high in renal tubular cells and is moderately expressed by a variety of tissues and cell types. Antibodies are typically produced by B cells that mature in the germinal center of the spleen and other lymphoid organs. Therapeutic production of antibodies with high levels of antibody-dependent cell-mediated cytotoxicity (ADCC) is determined by the content of GDP-D-mannose-4, 6-dehydratase (GMD), which can place N-acetylglucosamine (GlcNac) at the bisecting position of antibodies of the IgGl subtype present in the production cell line (J Biol Chem.), Vol.273, p.14582-. The production of antibodies with high ADCC activity using CHO-producing cell lines is not always optimal (J. Biochem 278:3466-3473, 2003). The present invention provides novel mammalian cell lines in order to obtain optimal ADCC activity of the produced antibodies, fusion proteins and cytotoxic biologics.

Transcription factors known to be associated with high levels of biological production can be co-transfected with the gene of interest to optimize the expression levels of the patient-specific cell line. For example, high levels of Pit-1 expression may cause high prolactin expression in a cell type, while blocking or preventing growth hormone expression (Genes Dev), 3: 946-9581989).

Monoclonal antibody production can be enhanced by optimizing gene codons using systems such as those developed by the people's Republic of China (Peoples Republic of China) seikagaku biotechnology Inc (nano Biological Inc), morphogenesis (morphinics) (proces of the national academy of sciences usa (Proc Natl Acad Sci), 103: 3557-.

Generation of synthetically produced pluripotent stem cells

Any type of synthetically produced pluripotent stem cell may be used to produce the personalized biological products of the invention. Two major classes are induced or reprogrammed pluripotent stem cells (ipscs) and stem cells produced by nuclear transfer (SCNT), ANT-OAR, and parthenogenesis.

BodyCell Nuclear Transfer (SCNT)Is a technique in which a non-nucleated egg is injected into the nucleus of an adult somatic cell and implanted into the uterus of a prepared recipient, resulting in live birth to obtain a complete nuclear gene clone. In addition, pluripotent stem cells have been derived from SCNT methods in culture (Cell reprogramming) 12:105-113,2010 and genomic research (Genome Res.) 19:2193-2201, 2009).

ModifiedNuclear transfer oocyte assisted reprogramming(ANT-OAR) is a similar technique to SCNT, however, the donor nuclei are prior to injection into recipient eggsGenetically altered to prevent ANT-oocytes from differentiating and forming whole organisms (genome study 19: 2193-.

Parthenogenesis (PGA) is also used to generate pluripotent stem cells using techniques such as zona-translucency nuclear transfer (zona-nuclear transfer), parthenogenetic activation; and cloning techniques such as SCNT and Parthenogenesis (PGA) have also been used to generate reprogrammed pluripotent stem cells (cell reprogramming, 12:105-113,2010 and Nature, 450: 497-5022007).

These pluripotent stem cells can be maintained in culture for a somewhat prolonged period of time, making them a potential source of biological production (e.g., recombinant proteins, DNA, and viruses).

Inducible or reprogramming spPSCs

Induced pluripotent stem cells are similar to natural pluripotent stem cells (e.g., Embryonic Stem (ES) cells) in many ways, such as expression of certain stem cell genes and proteins, chromatin methylation patterns, doubling times, embryoid body formation, teratoma formation, viable chimera formation, and potency and differentiability.

Induced pluripotent Stem Cells (often abbreviated as iPS Cells or ipscs) are a class of pluripotent Stem Cells (Nature Reports Stem Cells) 2007 artificially derived from non-pluripotent Cells (usually adult somatic Cells) by inducing "forced" expression of specific genes.

Induced pluripotent stem cells (ipscs) are generated by transfecting certain stem cell-associated genes into non-pluripotent cells. Induced pluripotent stem cells are typically derived by transfecting certain stem cell-associated genes into non-pluripotent cells such as adult fibroblasts. Transfection is usually accomplished by viral vectors (e.g., retroviruses or retrotransposons). The transfected genes included the major transcriptional regulators Oct-3/4 (Pou 5f 1) and Sox2, although other genes have been proposed to enhance induction efficiency. After 3-4 weeks, a small number of transfected cells begin to become morphologically and biochemically similar to pluripotent stem cells and are usually isolated by morphological selection, doubling time, or by reporter gene and antibiotic selection.

Embryonic stem cell-derived fibroblasts and adult fibroblasts, as well as other cells, have been reprogrammed to a pluripotent state by: fusion with embryonic Stem Cells (Cell.) 126: 652-. The induction of pluripotency can also be achieved by: modifying the methylation or polyadenylation state of the genome (scientific public library, integrated (PLoS One.), 4: e8419,2009); microRNA (developmental biology (Dev Biol.), 344:16-25,2010), small molecule activators of desired transcription factors, epigenetic reprogramming (Regen Med., 2:795-816, 2007); protein-based reprogramming (Blood 116: 386-; adding a culture supernatant or cell extract of the pluripotent cells in the culture; chemical or radiation or other means of gene mutation to reactivate pluripotency genes; and adding a growth factor or cytokine or cell signaling moiety that induces or maintains an endogenous pluripotent state.

The use of retroviruses to reprogram cells to a pluripotent state presents the risk of restoring immunodeficiency to gene therapy trials. Excision technology (such as Cre-lox) has been used to eliminate retroviruses after successful reprogramming, and the pagebeck (piggyBac) transposon method completely eliminates the need for retroviruses (biotechnological new (Curr Opin Biotechnol), 20: 516-.

Human ipscs have been generated by converting human fibroblasts into pluripotent stem cells using four key genes (Oct 3/4, Sox2, Klf4, and c-Myc) and a retrovirus system. Human ipscs have also been produced using the lentivirus system using OCT4, SOX2, NANOG and the different gene LIN 28. Adenovirus has also been used to transport the necessary four genes into the DNA of mouse skin and liver cells, thereby producing cells consistent with embryonic stem cells (Science 322(5903):945 949, 2008). Reprogramming of adult cells to ipscs can also be achieved by plastids in the absence of any viral transfection system at all (science 322(5903): 949-. Ipscs have been produced using pagebeck transposome systems, minicircle technology, protein stimulation or conditioned medium stimulation reprogramming.

The production of iPS cells is critically dependent on the genes used for induction. Oct-3/4 and certain members of the Sox gene family (Sox 1, Sox2, Sox3, and Sox 15) have been identified as key transcriptional regulators involved in the induction process, the absence of which would make induction impossible. However, other genes including certain members of the Klf family (Klf 1, Klf2, Klf4, and Klf 5), certain members of the Myc family (C-Myc, L-Myc, and N-Myc), Nanog, and LIN28 have been identified to increase induction efficiency.

Oct-3/4 (Pou 5f 1) (cDNA, available from Bioclone, San Diego CA) 20 (Nucleic Acids Res.) 17 (4613-20, 1992): oct-3/4 is a member of the octamer ("Oct") family of transcription factors and plays a key role in maintaining pluripotency. Oct-3/4⁺The absence of Oct-3/4 in cells such as blastomeres and embryonic stem cells causes spontaneous trophoblast differentiation, and thus the presence of Oct-3/4 results in the pluripotency and differentiation potential of embryonic stem cells. Several other genes in the "Oct" family (including Oct1 and Oct6, close relatives of Oct-3/4) failed to trigger induction, thus confirming the exclusivity of Oct-3/4 for the induction process.

The Sox family: like Oct-3/4, the Sox family gene is associated with maintaining pluripotency, but it is associated with multipotent and unipotent stem cells, in contrast to Oct-3/4, which is expressed only in pluripotent stem cells (developmental biology 227(2): 239-. Although Sox2 (cDNA, available from san diego bayer ohron, california) is the starting gene for induction (mammalian genome (mamm. genome) 5(10): 640-. Sox1 (cDNA, available from san diego bayer ohron, ca) produced iPS cells with similar efficiency as Sox2, and genes Sox3 (human cDNA, available from san diego bayer ohron, ca), Sox15, and Sox18 also produced iPS cells, although the efficiency was reduced.

Klf family: klf4 in the Klf family of genes is a factor for producing mouse iPS cells. Klf2 (cDNA, available from san diego bayer ohron, ca) and Klf4 (cDNA, available from san diego bayer ohron, ca) are factors capable of producing iPS cells, and the same is true for the relevant genes Klf1 (cDNA, available from san diego bayer ohron, ca) and Klf5 (cDNA, available from san diego bayer ohron, ca), although the efficiency is reduced.

The Myc family: the Myc family of genes are proto-oncogenes involved in cancer. C-myc (cDNA, available from san Diego Baker, Calif.) is a factor involved in the production of mouse iPS cells. However, c-myc may be unnecessary for the generation of human iPS cells. The use of "myc" family genes to induce iPS cells is problematic for the possibility of iPS cells as a clinical therapy, as 25% of mice transplanted with c-myc-induced iPS cells form lethal teratomas. N-myc (cDNA, available from san Diego Babyakron, Calif.) and L-myc have been identified as being inducible with similar efficiency in place of c-myc.

Nanog: (cDNA, available from san Diego Baker Oklung, Calif.) in embryonic stem cells Nanog along with Oct-3/4 and Sox2 are necessary to promote pluripotency (cell 113(5): 643-.

LIN 28: (cDNA, available from san Diego Baker, Calif.) LIN28 is an mRNA binding protein associated with differentiation and proliferation expressed in embryonic stem cells and embryonic cancer cells (developmental biology 258(2): 432-.

Identity of synthetically produced pluripotent stem cells

The spPSC produced is clearly similar to naturally isolated pluripotent stem cells (e.g., mouse and human Embryonic Stem Cells (ESCs), mESC and hESC, respectively) in the following respects, thus confirming the identity, authenticity and pluripotency of spPSC against naturally isolated pluripotent stem cells:

biological properties of cells:

the shape of the ring: ipscs are similar in morphology to ESCs. Each cell has a circular shape, a large nucleus and less cytoplasm. The population of ipscs is also similar to the population of ESCs. Human ipscs formed a well-defined, flat, close-packed colony similar to hescs and mouse ipscs formed a colony similar to mESC, an uneven and more aggregated colony compared to hESC.

Growth properties: doubling time and mitotic activity are the basis for ESCs, since stem cells must self-renew as part of their definition. ipscs are mitotically active, actively self-renewing, proliferating and dividing at a rate equal to ESC.

Stem cell markers: ipscs express the same cell surface antigen marker expressed on ESCs. Human iPSC expression markers specific for hESC, including SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, and Nanog. Mouse iPSCs, like mESCs, express SSEA-1 but not SSEA-3 and SSEA-4.

Stem cell gene: ipscs express genes expressed in undifferentiated ESCs including Oct-3/4, Sox2, Nanog, GDF3, REX1, FGF4, ESG1, DPPA2, DPPA4, and hTERT.

Telomerase activity: telomerase is necessary to maintain cell division independent of the Hayflick limit (Hayflick limit) which is about 50 cell divisions. hescs express high telomerase activity to maintain self renewal and proliferation, and ipscs also exhibit high telomerase activity and express hTERT (human telomerase reverse transcriptase), an essential component in the telomerase protein complex.

The pluripotency is as follows: ipscs can differentiate into fully differentiated tissues in a similar manner to ESCs:

differentiation of nerves: ipscs can differentiate into neurons expressing β III-tubulin, tyrosine hydroxylase, AADC, DAT, ChAT, LMX1B and MAP 2. The presence of catecholamine-associated enzymes may indicate that ipscs (e.g., hescs) may differentiate into dopaminergic neurons. Stem cell-associated genes are down-regulated after differentiation.

Heart differentiation: ipscs can differentiate into cardiomyocytes that spontaneously begin beating. Cardiomyocytes express TnTc, MEF2C, MYL2A, MYHC β, and NKX 2.5. Stem cell-associated genes are down-regulated after differentiation.

Teratoma formation: ipscs injected into immunodeficient mice spontaneously form teratomas after nine weeks. Teratomas are tumors containing multiple lineages of tissue derived from the three germ layers (endoderm, mesoderm, and ectoderm); this is in contrast to other tumors that typically have only one cell type. Teratoma formation is a hallmark test for pluripotency.

O embryoid body: hescs in culture spontaneously form globular embryo-like structures called "embryoid bodies" consisting of mitotically active and in the core of differentiated hescs and the periphery of cells that are fully differentiated from all three germ layers. ipscs also form embryoid bodies and have peripheral differentiated cells.

Tetraploid complementation: iPS cells from mouse fetal fibroblasts injected into quadruped blastocysts (which themselves can only form extra-embryonic tissues) can form intact non-chimeric fertile mice, although with low success rates. Tetraploid complementation analysis is a biological technique in which cells of two mammalian embryos are combined to form a new embryo. It is used to construct genetically modified organisms to study the consequences of certain mutations in embryonic development and in the study of pluripotent stem cells.

Induced pluripotent Stem Cells (S) have been generated from gut mesenteric Cells (reprogramming of Cells, 12: 237-. iPS cells can be generated theoretically from any cell type, but all 220 cell types in humans have not been studied systematically. Several recent studies have demonstrated that iPS cells retain 'memory' with respect to their original cell type. This is reflected in preferential (sometimes spontaneous, easiest) redifferentiation of iPS cells in culture towards their original cell type.

Isolation of endogenous Stem cells

Stem cells, including endogenous pluripotent stem cells (epscs), can be characterized and isolated by specific antigens expressed on their surface. Pluripotent stem cells can be characterized by the expression of Stage Specific Embryonic Antigen (SSEA), the transcription factors Oct4 and Nanog, as well as other markers, among other methods. The main type of endogenous pluripotent stem cells that have been isolated to date are very small embryonic-like (VSEL) stem cells.

VSELs are small (3-5 microns in diameter in mice and 3-7 microns in diameter for humans) and the ratio of nucleus to cytoplasm is high. VSELs were positive for SSEA1, Oct4, Nanog, Rex1, and other pluripotent stem cell markers and for CD133, CD34, AP, cMet, LIF-R, and CXCR 4. (J Am Coll Cardiol) 53(1):10-20,2009; Stem Cell review (Stem Cell Rev) 4:89-99,2008). They were negative for CD 45. VSELs are smaller than HSCs (3-6 vs 6-8 μm) and have higher nuclear/cytoplasmic ratios. The VSEL nucleus is large, contains open chromatin and is surrounded by a narrow cytoplasmic border with numerous mitochondria. Thus, their morphology is consistent with the original PSC.

The absolute number of circulating VSELs in PB is exceptionally low (1 to 2 cells in 1mL of blood under steady state conditions) and therefore special flow cytometry protocols must be applied for their identification and separation. Phenotypic markers used to identify VSELs include negative expression of CD45 (mouse and human), positive expression of Sca-1 (mouse), CXCR4, CD133, and CD34 (mouse and human), positive for stem cell markers (i.e., Oct-4, Nanog, and SSEA), and expression of several markers characteristic of ectodermal/germline stem cells.

Sorting of all VSELs present in 100mL UCB could be completed within 4 working days using only fluorescence activated cell sorter separation of VSELs. A more efficient and economical three-step fractionation protocol allows Lin-/CD45- & ltwbr/& gtThe recovery of UCB-VSEL was 60% of the initial number. The scheme comprises the steps of dissolving red blood cells in a low-tonicity ammonium chloride solution and performing the dissolving by using immunomagnetic beadsCell selection and Lin-/CD 45-based preservation by FACS using a size-labeled bead controlAnd (4) sorting the cells. The isolated cells were highly enriched in small highly original Lin-/CD45- & ltwbr/& gtOf cellsAndand (4) clustering.

Another method for sorting VSELs is based on the presence of several surface markers and the diameter of the cells. Briefly, the initial step is to lyse the red blood cells to obtain a fraction of nucleated cells. Erythrocyte lysis buffer was used instead of Ficoll centrifugation (Ficoll centrifugation) because the latter method may use up a very small population of cells. Cells were then stained with antibodies against Sca-1 (murine VSEL) or CD133 (human VSEL), total hematopoietic lineage antigen (CD 45), hematopoietic lineage markers (lin), and CXCR4 and sorted using a multi-parameter live sterile cell sorting system (MoFlo, Beckman Coulter; FACSAria, bedi medicine (beckton dickinson)). This method uses an "expanded lymphocyte gate" to include cases of 2-10 μm in diameter, including about 95% VSELs.

Endogenous stem cells may be contained in a mononuclear cell fraction from bone marrow, whole blood, cord blood, adipose tissue, or other sources, or they may be purified by selection for CD34, CD133, CD105, CD117, SSEA1-4, dye exclusion, or other specific stem cell antigens. Stem cells can be isolated by density gradient centrifugation using polysucrose-sodium diatrizoate (Ficoll-Hypaque) or other commercially available gradients from whole blood, bone marrow, cord blood, adipose tissue, tissue scrapings from olfactory mucosa and other sources of stem cells that can dissociate into single cell suspensions, such as umbilical cord tissue. Stem cells can be recovered from the mononuclear cell fraction produced by such procedures. Alternatively, Stem Cells can be found in other parts after density gradient centrifugation (Stem cell development (Stem Cells Dev.) 2011[ electronic version before printing ]). For example, cord blood may be diluted 1:1 in PBS, carefully spread onto a hestaupick (Histopaque) 1077 (Sigma) and centrifuged at 1500rpm for 30 minutes at room temperature. The resulting layers as depicted can be further processed for stem cell isolation. Layer 1 is a platelet layer, layer 2 is a leukocyte layer containing monocytes, layer 3 is a sucrose layer, and layer 4 is a red blood cell pellet layer also containing VSELs. Layers 1,2 and 3 can be collected, diluted with an appropriate medium (e.g., DMEM F12) in the presence or absence of FBS and centrifuged again to obtain a cell pellet. Layer 4 may be diluted with an appropriate medium (e.g., DMEM F12) and centrifuged at 800rpm for 15 minutes at room temperature in a standard tabletop centrifuge. Stem cells can be recovered primarily from layers 2 (buffy coat) and 4 (RBC pellet) according to the types of flow cytometry methods outlined above. FIG. 1 shows a typical view of the layers resulting from gradient centrifugation of whole blood. 1 displaying platelets; 2 displaying a buffy coat with MNCs and stem cells; 3 displaying the polysucrose; and 4 display RBC pellets and stem cells.

In addition, since glycosylation patterns and other post-translational modifications may differ between different tissues and cell types, patient-specific stem cell lines can be prepared from adult or somatic cells isolated from organs or cell types endogenously expressing biologics. See Repette-Demetz E (Rajpert-De Meyts E) et al, "morphological Changes in simple mucin-type O-glycans and polypeptide GalNAc transferase in human testis and testicular neoplasms are associated with germ cell maturation and tumor differentiation (Changes in the profile of simple mucin-type O-glycans and lipolytica GalNAc-transferases in human testae and telematic neoplasms) and with germ cell maturation and tumor differentiation, Vigorse literature (Vihorws Arch), Vol.451: 805-814 (2007). See Pevalova M. (Pevalova M.), et al, "Post-translational modifications of tau protein", journal of the Branisl Lek Listy, 107: 346-.

Drawings

Preferred and alternative examples of the present invention are described in detail below with reference to the following drawings:

figure 1 depicts the layers resulting from gradient centrifugation of whole blood.

Detailed Description

Production of immortalized spPSC and ePSC

In a preferred embodiment of the invention, spPSC and ePSC are preferably immortalized by non-viral induction of the larger T-antigen, typically using the polyomavirus monkey virus 40 (SV 40). See Ross MR, (Rose, MR) et al, (1983.) Expression of the larger T Protein of the Polyoma Virus Promotes establishment of "Normal" Rodent Fibroblast Cell Lines in Culture (Expression of the Large T Protein of Polyoma Virus proteins in Culture of "Normal" Rodent Fibroplast Cell Lines ". A.Natl.Acad.Sci.USA (PNAS) 80:4354-4358 (1983); and Hofmann M.C (Hofmann, M.C.) et al, "Immortalization of germ and somatic testis cells using the SV40large T antigen using SV 40" Experimental cell Research (Experimental cell Research), 201: 417-.

Overview of the examples Using synthetically generated, preferably induced pluripotent Stem cells or more preferably isolated endogenous pluripotent Stem cells

In a preferred embodiment:

1. isolating endogenous pluripotent stem cells;

2. immortalizing the ePSC;

3. transfecting immortalized ePSCs with a gene, virus or nucleic acid of interest using non-viral techniques;

4. subsequently inducing the transfected immortalised ePSCs to differentiate into germ line cells, preferably ovarian cells, so as to be able to express the nucleic acid product in a more efficient manner;

5. differentiated cells can now be induced to express a nucleic acid product of interest from a previously transfected vector containing the nucleic acid of interest.

In another preferred embodiment:

1. isolating somatic cells;

2. transforming somatic cells into induced pluripotent stem cells (iPS cells);

3. immortalizing the iPS cells;

4. transfecting an immortalized iPS cell with a vector containing a gene, virus or nucleic acid of interest;

5. then inducing the transfected immortalized iPS cells to redifferentiate into adult cells so as to express proteins in a more efficient manner;

6. the redifferentiated cells can now be induced to express the protein of interest from a previously transfected vector containing the gene of interest.

In an even more preferred embodiment:

1. isolating endogenous pluripotent stem cells;

2. transfecting immortalized ePSCs with a gene, virus or nucleic acid of interest using non-viral techniques;

3. subsequently inducing the transfected immortalised ePSCs to differentiate into germ line cells, preferably ovarian cells, so as to be able to express the nucleic acid product in a more efficient manner;

4. differentiated cells can now be induced to express a nucleic acid product of interest from a previously transfected vector containing the nucleic acid of interest.

Methods of implementing preferred embodiments of the present invention are well known to those skilled in the art.

In alternative embodiments, the ePSC or spPSC can be transfected with a vector containing a nucleic acid of interest prior to immortalization. See Du C. (Du C.) et al, "production of Variable and Fixed Length sirnas from a novel siRNA expression vector" (generation of Variable and Fixed Length siRNA expression vector), biochem (Biomed. & biophysis. res. comm.) 545:99-105 (2006); zhuyork (York Zhu), U.S. patent application No. 12/313,554, filed on 21/11/2008. Alternatively, the ePSC or spPSC cells can be induced to redifferentiate, which can then be immortalized and the immortalized, redifferentiated cells can be transfected with a vector containing a nucleic acid of interest. Another possibility is that ePSC or spPSC cells can be induced to redifferentiate, that the redifferentiated cells can be transfected with a nucleic acid of interest and that the redifferentiated, transfected cells can be immortalized.

Preferably the ePSC or spPSC cells are expanded in culture, preferably in cell culture medium containing autologous human serum and stem cell factor or leukemia inhibitory factor, prior to redifferentiation.

The polypeptide or protein produced may be any polypeptide or protein. Of particular interest are polypeptides or proteins selected from the group consisting of: erythropoietin, factor VIII, factor IX, thrombin, antibodies or antibody fragments, interferon alpha 2A and 2B (see U.S. Pat. Nos. 4,810,645 and 4,874,702), interferon beta (see U.S. Pat. No. 4,738,931), consensus interferon (see U.S. Pat. No. 5,661,009), growth hormone, antihemophilic factor, G-CSF, GM-CSF, soluble receptor, fusion protein such as soluble receptor fused to immunoglobulin (Ig) constant region (see U.S. Pat. No. 5,155,027), TGF-beta, Bone Morphogenetic Protein (BMP), TGF alpha, interleukin 2, beta-glucocerebrosidase or analogs thereof, alpha 1-protease inhibitors, fibrin, fibrinogen, von Willebrand factor, imiglucerase (imiglucerase), galactosidase (agalsidase) beta, laronidase (laronidase), Arabinosidase alpha, thyrotropin alpha, and thymosin alpha.

Any antibody or antibody fragment can be produced according to the methods of the invention. Of particular interest are those antibodies or antibody fragments that bind to a target, wherein the target is selected from the group consisting of: tumor Necrosis Factor (TNF) molecules, growth factor receptors, Vascular Endothelial Growth Factor (VEGF) molecules, interleukin 1, interleukin 4, interleukin 6, interleukin 11, interleukin 12, interferon gamma, nuclear factor kappa-B receptor activator ligand (RANKL), and Blys.

Induction of stem cell differentiation

To optimize production of the protein of interest, transfected ePSC or spPSC cells should be induced to differentiate into adult cells.

In an alternative embodiment, a population of ePSC or spPSC cells can be expanded and differentiation can be induced, and the differentiated cells can then be transfected with a nucleic acid of interest. The stem cells can be induced to differentiate in culture to somatic cell types by: addition of various growth factors to the culture (blood, 85: 2414-. For example, retinoic acid, TGF- β, Bone Morphogenic Protein (BMP), ascorbic acid, and β -glycerophosphate cause osteoblast production; indomethacin (indomethacin), IBMX (3-isobutyl-1-methylxanthine), insulin and triiodothyronine (T3) cause the production of adipocytes; alpha FGF, beta FGF, vitamin D3, TNF-beta and retinoic acid cause the production of myocytes (heart-derived stem cells (CARDIAC DERIVED STEM CELLS) (WO/1999/049015) 3 months 1998). Germ cells have been produced from pluripotent stem cells using monolayer culture, forming Embryoid Bodies (EBs), co-aggregating with BMP 4-producing cells and using testis or ovary cell conditioned media, or forming EBs with recombinant human Bone Morphogenetic Proteins (BMPs) (scientific public library, integrated 2009;4(4): e 5338). Germ cell marker genes include PR domain containing protein 1 with ZNF domain (PRDM 1, also known as BLIMP 1), PR domain containing protein 14 (PRDM 14), protein arginine methyltransferase 5 (PRMT 5), DPPA3, IFITM3, GDF3, C-kit, chemokine (C-X-C motif) receptor 4 (CXCR 4), NANOS1-3, DAZL, VASA, PIWI family genes (PIWIL 1 and PIWIL2, respectively known as HIWI and HILI in humans), Mut-L homolog-1 (MLH 1), synaptonectin 1 (SCP 1), and SCP 3. The resulting germ cell line can be genetically modified to express a gene or protein product of interest similarly to the use of Chinese Hamster Ovary (CHO) cells and other currently used manufacturing cell lines. Differentiation strategies for obtaining multiple somatic cell lines from multiple stages of stem cells are well known to those skilled in the art of stem cell biology.

Transcription factors known to be associated with high levels of biological manufacturing can be co-transfected with the gene of interest to optimize expression levels in patient-specific cell lines. For example, high levels of Pit-1 expression may result in high prolactin expression in a cell type, while blocking or preventing growth hormone expression (Gene and development, 3:946-958, 1989).

Monoclonal antibody production can be enhanced by optimizing gene codons using systems such as those developed by seupian biotechnology limited, morphogenesis or other standard biotechnological methods.

According to the present invention, recombinant polypeptides and proteins are produced in epscs and sppscs, wherein sppscs and epscs are produced or isolated from cells of a particular species or population due to the fact that: some breeds or families of animals of a particular species have different glycosylation patterns in polypeptides or proteins produced by the particular breed or family. According to the present invention, a population is a population of members that are associated with each other by a common inheritance, which often consists of a common ancestor or a sibling (a convention of marrying within a particular population, such as Ashkenazi Jews). In general, it is a highly bioagent self-sustaining population. Examples of populations that may have different glycosylation patterns in polypeptides and proteins are found in Levinson (Levinson), David (1998), populations of countries of the world: a convenient Reference manual (Ethnic Groups Worldwide: A Ready Reference Handbook), Greenwood Publishing Group (Greenwood Publishing Group).

Methods and systems for stem cell therapy

The invention also includes a method for facilitating stem cell therapy without the formation of teratomas. The present invention teaches how to advantageously exploit the observed 'memory' of reprogrammed somatic cells, herein referred to as synthetically produced pluripotent stem cells (sppscs), as defined above, in order to obtain greater therapeutic benefit. The memory of sppscs gives preference to redifferentiation to the original cell type prior to reprogramming, providing a means to enhance the safety and therapeutic utility of sppscs for regenerative medicine.

General procedure

The invention relates to

1. Isolation of somatic cells of interest, particularly somatic cells that are desired to be regenerated;

2. somatic cell transformation into synthetically produced pluripotent stem cells (spPSC), particularly induced pluripotent stem cells (ipscs), as described above;

expansion of the spPSC population, wherein the spPSC maintains an intrinsic epigenetic memory of the somatic cell;

the spPSC is redifferentiated in culture into redifferentiated somatic cells having the original somatic cell type; and

5. administering or delivering the redifferentiated somatic cells to a region of the body in need of the cell type.

Several recent studies have demonstrated that spPSC cells retain 'memory' with respect to their original cell type (stem cells 28:1981-1991, 2010), (Nature, 467(7313):285-90, 2010), Nature Biotechnology (NatBiotechnol), 28:848-855, 2010) and (molecular human reproduction (Mol Hum Reprod.), 16:880-885, 2010). This is reflected in the preferential (sometimes spontaneous, easiest) redifferentiation of sppscs in culture towards their original cell type. Scientists and clinicians scientists have used tunnel assays to focus on generating pluripotent stem cells that may be suitable for clinical therapy, drug discovery, disease modeling, or toxicity screening (biotechnology new (Curr OpinBiotechnol), 20: 516-. Indeed, scientists disclosing data on the 'memory' aspect of reprogramming pluripotent stem cells have focused on the limitation of this property of spPSC cells with respect to treatment development, but have missed the importance of this property in providing therapeutic utility of these cells.

The memory aspects of sppscs have been observed by somatic cells of various origins. For example, lentiviral expression using OCT4, SOX2, LIN28, and Nanog reprograms primary fetal retinal epithelial cells to iPSC (Stem cells 28:1981-1991, 2010) and passes standard tests for pluripotency; they form teratomas and express pluripotent stem cell markers. Upon removal of basic FGF from the growth medium, some retinal spPSC cell lines spontaneously redifferentiate back to the retinal epithelial cell lineage. Approximately 60% of the cells spontaneously differentiated from human fetal retinal epithelial spPSC cells are retinal epithelial cells, which can be compared between 5% and 16% to retinal epithelial cells from human fetal lung fibroblasts, human foreskin fibroblasts, or human ESC spPSC cells. However, one out of every three spPSC cells from human fetal retinal epithelial cells failed to differentiate into retinal epithelial cells. Kim (Kim) et al (Nature, 467(7313):285-90, 2010) used retroviruses to introduce Oct4, Sox2, Klf4 and Myc to reprogram myeloid progenitors and dermal fibroblasts from aged mice. All stem cell lines they produce exhibit pluripotency using criteria that are generally applicable to human samples. Subsequent differentiation of their reprogrammed pluripotent stem cells demonstrates that hematopoietic sources are more readily redifferentiated towards the hematopoietic lineage than are fibroblast sources, and similarly, fibroblast sources are more readily redifferentiated towards the mesenchymal lineage than are hematopoietic sources. The authors also show that this propensity to redifferentiate, preferably towards their original somatic lineage, can be overcome in part by differentiating towards a hematopoietic lineage followed by another round of pluripotent reprogramming followed by differentiation. For example, reprogrammed cells derived from neural progenitor cells that differentiate to a hematopoietic lineage, followed by reprogramming to pluripotency, exhibit higher hematopoietic colony formation than neural progenitor cells that differentiate to neural cells, reprogram, and then differentiate to hematopoietic cells.

Similar observations that reprogrammed cells preferentially redifferentiate toward their original cell type have been obtained using non-viral reprogramming of human fetal neural progenitor cells (scientific public library-integrated, 4: e7076-e7088,2009). The resulting reprogrammed cells expressed several markers of pluripotency (markers for all three germ layers) and were able to form embryoid bodies and teratomas in culture, however, using gene chip analysis, the authors demonstrated that reprogrammed neural progenitor cells retained some expression of neural stem cell genes. Porro (Polo) et al (Nature Biotechnology, 28:848-855, 2010) derived pluripotent reprogramming cells from mouse tip derived fibroblasts, splenic B cells, myelogranulocytes, and skeletal muscle precursors. Spontaneous differentiation studies indicate that splenic B-cell and myeloid granulocyte reprogrammed spPSC cells produce hematopoietic progenitor cells more efficiently than fibroblast or skeletal muscle derived spPSC cells. Interestingly, serial passage of these multiple spPSC cell lines abolished gene and methylation differences at passage 16, and then the cells also displayed equal differentiation efficiency, unlike earlier results at passage 4. Interestingly, this phenomenon of differential differentiation potential is not limited to reprogrammed somatic cells, which has also been observed for embryonic stem cell lines that have been found to have different genetic profiles and spontaneously differentiate preferentially towards certain cell lineages (Nature Biotechnology, 26:313-315,2008) (human New Production (Hum Reprod Update), 13:103-120,2007) (developmental biology, 307:446-459,2007) (J. Cell. biol. Med., 10:44,2009).

Stem cells can be characterized and isolated by specific antigens expressed on their surface. Pluripotent stem cells can be characterized by the expression of Stage Specific Embryonic Antigen (SSEA), the transcription factors Oct4 and Nanog, as well as other markers, among other methods. Embryonic stem cell-derived fibroblasts and adult fibroblasts, as well as other cells, have been reprogrammed to a pluripotent state by: fusion with embryonic stem cells (cell 126: 652-. The induction of pluripotency can also be achieved by: modifying the methylation or polyadenylation state of the genome (scientific public library integrated 4: e8419,2009); microRNA (developmental biology, 344:16-25,2010), small molecule activation factors for desired transcription factors, epigenetic reprogramming (regenerative medicine, 2:795-816, 2007); protein-based reprogramming (blood 116:386-395, 2010); adding culture supernatant or cell extract of pluripotent cells in culture, chemical or radiation or other means of gene mutation to reactivate pluripotency genes, or adding growth factors or cytokines or cell signaling moieties that induce or maintain endogenous pluripotency states. The use of retroviruses to reprogram cells to a pluripotent state presents the risk of restoring immunodeficiency to gene therapy trials. Excision techniques (such as Cre-lox) or the pagebeck transposon method have been used to eliminate retroviruses after successful reprogramming (new biotechnology, 20: 516-. Cloning techniques (such as SCNT) and Parthenogenesis (PGA) (cell reprogramming, 12:105-113, 2010) have also been used to generate reprogrammed pluripotent stem cells (Nature, 450:497-502, 2007).

Significant resources (economic, intellectual and labor) have been invested in finding a variety of sources of pluripotent stem cells that would be primarily expected to be suitable for regenerative drug therapy in humans. Unfortunately, with the exception of adult VSELs (stem cell review 4:89-99,2008), all pluripotent stem cells isolated to date have been frustrated by the problems of teratoma formation, tumor formation, and even neoplasms. Therefore, in order to take advantage of the economic, intellectual and labor investments that have been made in the past 15 or so years, it is necessary to utilize these pluripotent stem cells to some extent.

The stem cells can be induced to differentiate in culture to somatic cell types by: addition of various growth factors to the culture (blood, 85: 2414-. For example, retinoic acid, TGF- β, Bone Morphogenic Protein (BMP), ascorbic acid, and β -glycerophosphate cause osteoblast production; indomethacin, IBMX (3-isobutyl-1-methylxanthine), insulin and triiodothyronine (T3) cause the production of adipocytes; aFGF, bFGF, vitamin D3, TNF-. beta.and retinoic acid caused the production of myocytes ((WO/1999/049015) 3 months 1998). Differentiation strategies for obtaining multiple somatic cell lines from multiple stages of stem cells are well known to those skilled in the art of stem cell biology.

Stem cell therapy is being investigated and ideally used to treat many human diseases. NIH websitewww.clinicaltrials.govThe clinical trial information contained in (a) lists over 3000 stem cell studies. Diseases under evaluation include: hematological malignancies, leukemias, lymphomas, cancer, osteopetrosis, aplastic anemia and cytopenia, sickle cell disease and thalassemia, limbal stem cell deficiencyBreast cancer, acute myocardial infarction, coronary artery disease, peripheral vascular disease, heart failure, type 1 diabetes, type 2 diabetes, stroke, spinal cord injury, neuroblastoma, multiple sclerosis, systemic sclerosis, lupus erythematosus, chronic wound healing, burns, fracture healing, cartilage repair, CNS tumors, osteoarthritis, renal failure, Parkinson's disease, myeloma, diabetic foot, cirrhosis and biliary cirrhosis, dilated cardiomyopathy, anemia, retinitis pigmentosa, crohn's disease, diabetic neuropathy, mastocytosis, ovarian cancer, epilepsy, myasthenia gravis, autoimmune disease, granulomatous disease, osteonecrosis, liver failure, PMD disease, lipodystrophy, demyelinating disease, cartilage defects, retinal disease, lupus nephritis, Alzheimer's disease, Traumatic brain injury, sarcoma, myositis, hyperglycemia, macular degeneration, ulcerative colitis, muscle degeneration and other diseases.

The stem cells can be isolated using a variety of markers known to those skilled in the art. For example, can be inhttp://stemcells.nih.gov/info/scireport/appendixe.asp#eiiA list of commonly used stem cell markers was found. Neural stem cells can be isolated by using CD 133; isolating mesenchymal stem cells and progenitor cells by using Bone Morphogenetic Protein Receptor (BMPR); isolation of hematopoietic stem cells by CD 34; isolating mesenchymal stem cells by a combination of CD34+ Sca1+ Lin-markers; isolating hematopoietic and mesenchymal stem cells by ckit, Stro1 or Thy 1; isolating neural and pancreatic progenitor cells by nestin; isolating ectodermal, neural, and pancreatic progenitor cells by vimentin; and other markers.

The invention further provides methods for safely reprogramming and redifferentiating somatic cells for regeneration of a drug. Suitable somatic cells may include fully differentiated somatic cells, progenitor cells, or more primitive stem cells. Depending on the organ to which regenerative therapy is required, more or less primitive stem cells can be obtained by organ puncture, biopsy, scrapings, or surgical retrieval. When it is possible to obtain these stem cells, it is preferable to use relatively primitive stem cells. Less preferred but preferred over fully differentiated somatic cells is the use of progenitor cells.

Isolation of somatic cells from specific organs as therapeutic targets, reprogramming those somatic cells, short-term expansion in culture to ensure maintenance of the inherent 'memory' of spPSC, followed by re-differentiation into primitive cell types in culture, followed by therapeutic application allows for a pluripotent stem cell approach to treat patients with reduced risk of tumor or teratoma formation.

Reprogrammed somatic cells can be used between passage 1 and passage 12, most preferably at passage 4. The reprogrammed somatic cells can be differentiated to the cell type for desired regeneration according to standard cell biology and differentiation techniques. The generated therapeutic cells can be administered by intravenous, intra-arterial, intramuscular, or other injection methods using standard injection techniques, which can include catheters, such as nogasar or MyoStar injection catheters or other approved catheter injection devices. Alternatively, therapeutic cells can be administered into the target tissue by minimally or more invasive surgical methods. The therapeutic cells may be administered in a buffer composition containing between 0% and 15%, most preferably 5% autologous human serum albumin. For the treatment of diseases of the central nervous system, autologous cerebrospinal fluid-buffered therapeutic cells will preferably be used. Therapeutic cells can also be administered by using scaffolds such as collagen, fibrinogen or other extracellular matrix or combination of extracellular matrices or by holding the cells in place and maintaining contact with the organ in need of regenerative repair using thin wires such as those produced using alginate or other standard methods.

Preferably, a minimum of 500 therapeutic cells will be administered, and typically cell therapy will utilize 1,500 to 5 million cells. More preferably, between 1,500 million and 1 million cells will be administered in order to obtain the best benefits.

As an example, for cell therapy for neurological diseases such as Alzheimer's Disease, Parkinson's Disease, stroke, Huntington's Disease, multiple sclerosis, paralysis and other diseases of the Central Nervous System (CNS), the olfactory mucosa (J Spinal Cord Cord Med.) 29:191-203,2006) will preferably be isolated using a hard endoscope as disclosed. Neural stem cells and progenitor cells or olfactory ensheathing cells are isolated from the olfactory mucosa according to methods well known to those skilled in the art of regenerative medicine. Alternatively, neural crest stem cells can be isolated from human hair follicles (Bionewspaper (Folia Biol.) 56:149-157, 2010). Most preferably, neural stem cells are reprogrammed by the addition of a defined factor, expanded and passaged for 4 passages, redifferentiated to a particular central nervous system cell type by the addition of a defined factor, and then administered to a patient for regenerative therapy.

The present invention provides methods for safely reprogramming and redifferentiating somatic cells for regeneration of a drug. Suitable somatic cells may include fully differentiated somatic cells, progenitor cells, or more primitive stem cells. Depending on the organ to which regenerative therapy is required, more or less primitive stem cells can be obtained by organ puncture, biopsy, scrapings, or surgical retrieval. When it is possible to obtain these stem cells, it is preferable to use relatively primitive stem cells. Less preferred but preferred over fully differentiated somatic cells is the use of progenitor cells.

For spinal injury repair, olfactory mucosa was removed by hard endoscope, neural stem cells were isolated using neurosphere test (science, 255: 1707-. The resulting neural stem cells were reprogrammed by adding episomal vectors for delivery of OCT4 and NANOG, and selected with hygromycin for 5-7 days. The cells are passaged and expanded up to passage 4 to obtain a non-integrated population, followed by differentiation to olfactory ensheaths and stem cell-like neural progenitor cells using autologous cerebrospinal fluid or defined factors. Damaged spinal cord is exposed during surgery using standard midline incision and posterior midline myelotomy, scar tissue is removed if allowed, therapeutic cells are buffered in autologous cerebrospinal fluid, seeded onto bioactive scaffolds and applied directly to the injured spinal cord.

Examples of the invention

Example 1

Genetically modifying synthetically produced patient-specific pluripotent stem cells to produce recombinant proteins

Genetically modified to induce biomanufacturing using synthetically produced pluripotent stem cells (sppscs) such as SCNT or PGA or ANT-OAR or iPSC from a patient. SCNT-derived stem cells are prepared by transferring the nucleus of a patient cell into an enucleated oocyte that has been prepared. ANT-OAR-derived stem cells are prepared by genetically modifying nuclear DNA of a patient and then transferring the modified nucleus into a prepared enucleated oocyte. iPSC-derived stem cells are prepared by reprogramming the cells of a patient using genetic modification, activation of pluripotent transcription factors, epigenetic modification or other methods known in the art as described above. The resulting synthetically generated patient-specific stem cell lines are 'banked' in the form of master cell banks and working banks for subsequent genetic modification for biological fabrication.

Germ cells are generated from pluripotent stem cells using monolayer culture, forming Embryoid Bodies (EBs), co-aggregating with BMP4 producing cells, using testicular or ovarian cell conditioned media, or forming EBs with recombinant human Bone Morphogenic Protein (BMP). Germ cells are identified by expression of marker genes that may include PR domain-containing protein 1 with ZNF domain (PRDM 1, also known as blip 1), PR domain-containing protein 14 (PRDM 14), protein arginine methyltransferase 5 (PRMT 5), DPPA3, IFITM3, GDF3, C-kit, chemokine (C-X-C motif) receptor 4 (CXCR 4), nans 1-3, DAZL, VASA, PIWI family genes (PIWIL 1 and PIWIL2, respectively known as HIWI and HILI in humans), Mut-L homolog-1 (MLH 1), synaptonectin 1 (SCP 1), and SCP 3. The resulting germ cells are transfected with the gene of interest (e.g., factor VIII) according to methods commonly used to make recombinant factor VIII.

Example 2

Genetically modifying synthetically produced patient-specific pluripotent stem cells to produce recombinant insulin

Synthetically produced pluripotent stem cells (sppscs) such as SCNT or PGA or ANT-OAR or iPSC-derived stem cells from patients are used for genetic modification to induce biomanufacturing. SCNT-derived stem cells are prepared by transferring the nucleus of a patient cell into an enucleated oocyte that has been prepared. ANT-OAR-derived stem cells are prepared by genetically modifying nuclear DNA of a patient and then transferring the modified nucleus into a prepared enucleated oocyte. iPSC-derived stem cells are prepared by reprogramming the cells of a patient using genetic modification, activation of pluripotent transcription factors, epigenetic modification or other methods known in the art as described above. The resulting synthetically generated patient-specific stem cell lines are 'banked' in the form of master cell banks and working banks for subsequent genetic modification for biological fabrication.

According to the general use for Engineering enhanced protein secretion expression in insulin-applied yeasts (Engineering-enhanced protein secretion expression in yeast with application to insulin) in s.cerevisiae (s.cerevisiae) [ kersen T. (Kjeldsen T.) "et al, J.Biochem., 277:18245-18248 (5.2002); zhang B et al, "Intracellular retention of newly synthesized insulin in yeast is caused by endoproteolytic processing in the Golgi complex" (J.Cell. Biol., 153:1187-1198 (6 months 2001)); and Cristenssen C. (Kristensen C.), et al, "Alanine scanning mutagenesis of insulin" (Alanine scanning mutagenesis of insulin), "J. biochem., 272:12978-12983 (5 months 1997) ] or E.coli [ Sun YJ. (SonYJ.)" et al, "influence of beta-mercaptoethanol and hydrogen peroxide on the enzymatic conversion of human proinsulin to insulin" (Effects of beta-mercaptoethanol and hydrogen peroxide on human proinsulin to insulin), "J. Microbiol. Biotech., 18:983-989 (5 months 2008) ] methods for the production of insulin for expression of insulin precursors in patient-specific dry cells followed by processing and purification according to standard methods. Insulin precursors expressing patient-specific cell lines are 'banked' as master and working cell banks for subsequent insulin production.

Example 3

Production of beta cells for insulin production using genetic modification of synthetically produced patient-specific pluripotent stem cells

To produce insulin, somatic cells from a patient are used for genetic modification to generate sppscs and these sppscs are used to induce biological manufacturing. spPSC-derived stem cells are prepared by reprogramming the cells of a patient using genetic modification, activation of a pluripotent transcription factor, epigenetic modification, or other methods known in the art. The resulting patient-specific stem cell lines are 'banked' in the form of master cell banks and working banks for subsequent genetic modification for biological fabrication. Alternatively, endogenous pluripotent stem cells (ePSCs) may be isolated and pooled according to the techniques described above.

Expression of insulin precursors in patient-specific stem cells is performed according to the methods generally used for the production of insulin in Saccharomyces cerevisiae or Escherichia coli as described above. Following transfection with the appropriate insulin Gene construct Gene, as can be seen in the beta cell biology Unionhttp://www.protocolonline.Org/prot/Cell_Biology/Stem_Cells/DifferentiatioHtml. Online scheme (Protocol Online.) [ web ] of _ Stem _ Cell/index][ quote: 2010, 12 months and 19 days]The standard protocol in the following literature allows the differentiation of the cells towards the beta cell lineage: [ Shi Y. (Shi, Y.), et al "induced differentiation of embryonic stem cells into pancreatic beta cells (induced embryonic stem cells to differential intercurrent pancreatic beta cells by a novel three-step method using activin A and all-trans retinoic acid" stem cells, 23: 656-; or lithotripteryl K. (Tateishi, K.) "Generation of insulin-secreting islet-like clusters from human skin fibroblasts" (Generation of insulin-secreting islets-like clusters), J.Biochem., 283:31601-]。

The resulting expressed biological product is then processed and purified according to standard methods. The generated patient-specific stem cell lines expressing insulin precursors are 'banked' in the form of master cell banks and working banks for subsequent genetic modification for biological manufacturing.

Alternatively, the resulting patient-specific stem cells are differentiated towards the beta cell lineage according to protocols that are standard in History et al (supra) or lithology et al (supra) of the beta cell biology alliance (supra). Once differentiated, expression of insulin precursors in patient-specific stem cells is performed according to the methods generally used for the production of insulin in saccharomyces cerevisiae or escherichia coli as discussed above. The resulting expressed biological product is then processed and purified according to standard methods.

Example 4

Isolation of antibody-producing adult (somatic) cells for reprogramming and transfection to produce biological antibody therapeutics

Antibody-producing B cells are isolated from peripheral blood, bone marrow, and other readily available hematopoietic cell sources for the purpose of generating patient-specific production cell lines to produce therapeutic antibody biologics. B cells were isolated using available kits based on CD19 expression (stem cell Technologies). Limiting dilution or cell sorting methods may be employed to select cells that produce the highest levels of immunoglobulin (Ig). After a brief expansion, clonal, high Ig-producing cells are reprogrammed to a pluripotent or progenitor state using standard reprogramming techniques. The resulting patient-specific stem cells are transduced with the desired antibody gene construct using standard molecular biology techniques and methods. The resulting expressed antibody therapeutics are processed and purified according to currently advanced biotechnological methods, whether publicly available or privately held by the owner of the combination of substances for the antibody therapeutics. Methods for obtaining the desired purified antibody product include ion exchange chromatography.

Example 5

Isolation of antibody-producing adult (somatic) cells for reprogramming and transfection to redifferentiate into antibody-producing somatic cells Producing a biological antibody therapeutic agent

Antibody-producing B cells are isolated from peripheral blood, bone marrow, and other readily available hematopoietic cell sources for the purpose of generating patient-specific production cell lines to produce therapeutic antibody biologics. Limiting dilution or cell sorting methods may be employed to select cells that produce the highest levels of immunoglobulin (Ig). After a brief expansion, clonal, high Ig-producing cells are reprogrammed to a pluripotent or progenitor state using standard reprogramming techniques. The resulting patient-specific stem cells are transduced with the desired antibody gene construct using standard techniques and methods. Following genetic modification, B cell maturation is promoted by activation of TLR4 in CD40L, BAFF, toll-like receptor activation (TLR) [ forest e.a. (Hayashi e.a.): independently and in cooperation with B lymphocyte activating factors (TLR 4 proteins Bcell activation factor), "J Immunol.," 184:4662-4672(2010) ] or other B Cell maturation factors known in the art (such as B Cell Receptor (BCR) activation and activation of the notch receptor ligand family [ Palanich. A. (Palanichamamy. A.) "et al" novel human transitional B Cell populations revealed by B Cell depletion therapy (Novelhuth Cell transformation B cells promoters recovery strategy), 2006, J. 182, page 5982-5993 (2009); Thomas M.D. (Thomas M.D.) "human peripheral cells maturation-mediated cells of No. 239 cells"; Cell maturation of B cells) can be produced by the immunological culture of multiple cells in a patient (Cell maturation line) 92).

The potency and affinity of therapeutic antibodies can be increased by using the methods of morphogenesis techniques described in, for example, Li J. (Li J.) et al, "production of Human antibodies for immunotherapy development by Human B-cell hybridoma technology", Proc. Natl.Acad.Sci.USA, 103:3557-3562 (2006). The resulting expressed antibody therapeutics are processed and purified according to currently advanced biotechnological methods (whether publicly available or privately held by the owner of the combination of substances for the antibody therapeutics).

Alternatively, patient-specific pluripotent cell lines are differentiated into mature antibody-producing B cells by culturing in the presence of CD40L, BAFF, toll-like receptor activation (TLR) (see forest et al, supra), or other B cell maturation factors as known in the art, such as B Cell Receptor (BCR) activation and activation of the scored receptor ligand family (see pananichmi a. et al, supra and thomas m.d. et al, supra). The potency and affinity of therapeutic antibodies can be increased by methods using morphogenetic techniques as described (see Li et al (supra)).

The resulting antibody-producing patient-specific cells are transduced with the desired antibody gene construct using standard techniques and methods. The resulting expressed antibody therapeutics are processed and purified according to currently advanced biotechnological methods (whether publicly available or privately held by the owner of the combination of substances for the antibody therapeutics).

Example 6

Generation of patient-specific cell lines for the production of high-activity ADCC antibodies

Post-translational modification of N-acetylglucosamine (GlcNac) of immunoglobulins is important for antibody-dependent cell-mediated cytotoxicity (ADCC), and Non-fucosylated GlcNac residues have the highest affinity for Fc γ receptor (Morie K.) et al, "Non-fucosylated therapeutic antibodies: next-generation therapeutic antibodies" (Non-glycosylated therapeutic antibodies: the next generation therapeutic antibodies), "Cytotechnology (Cytotechnology), 55:109-114 (2007)).

Thus, when antibody therapeutics with high levels of ADCC are desired, patient-specific cell lines capable of transferring appropriate levels of GlcNac and rendering GlcNac non-fucosylated are desired. Cancer cells are known to express higher levels of GMD, and therefore, since cancer stem cells and pluripotent cells have similar gene characteristics, it is suspected that pluripotent cells may also express high levels of GMD, an enzyme responsible for post-translational GlcNac ligation. In normal tissues, colon and pancreas express the highest levels of GMD. For the manufacture of therapeutic antibodies whose efficacy depends on ADCC activity, such as Rituximab (Rituximab) or Herceptin (Herceptin), the lack of FUT8 responsible for the enzymes that fucosylate the antibody in patient-specific stem cell lines would be desirable. For example, the ADCC activity of a monoclonal antibody produced in rat hybridoma YB2/0 cells was 50-fold greater than that of the same monoclonal antibody produced using CHO cells. Adipose-derived stem and germ cell lines and B-cell lymphomas express higher than average FUT8, while Hematopoietic Stem Cells (HSCs), immature B cells, normal skeletal muscle express lower than average FUT 8.

Hematopoietic stem cells were isolated from bone marrow aspirate or from whole blood apheresis according to standard methods with or without prior treatment with a stem cell mobilizing agent. The isolated HSCs are then reprogrammed to pluripotency as previously described. The resulting patient-specific stem cells are transduced with the desired antibody gene construct using standard techniques and methods. Following gene transfection, patient-specific pluripotent cell lines are differentiated into mature antibody-producing B cells by culturing in the presence of CD40L, BAFF, toll-like receptor activation (TLR) (see forest e.a. et al (supra)), or other B cell maturation factors as known in the art, such as B Cell Receptor (BCR) activation and activation of the scored receptor ligand family (see pananicami a. et al (supra) and tomassas m.d. et al (supra)). The potency and affinity of therapeutic antibodies can be increased by methods using morphogenetic techniques as described (see plum et al (supra)). The resulting expressed antibody therapeutics are processed and purified according to currently advanced biotechnological methods, whether publicly available or privately held by the owner of the substance combination with respect to the antibody therapeutic.

Alternatively, antibody-producing B cells are isolated from peripheral blood, bone marrow, and other readily available hematopoietic cell sources for the purpose of generating patient-specific production cell lines to produce therapeutic antibody biologics. Limiting dilution or cell sorting methods may be used to select the cells that produce the highest levels of immunoglobulin (Ig). After a brief expansion, clonal, high Ig-producing cells are reprogrammed to a pluripotent or progenitor state using standard reprogramming techniques. The resulting patient-specific stem cells are transduced with the desired antibody gene construct using standard techniques and methods. Following genetic modification, the patient-specific pluripotent cell line is differentiated towards HSCs, immature B cells or skeletal muscle cells in order to produce therapeutic antibodies that are low or deficient in trehalose.

While the preferred embodiments of the invention have been illustrated and described, as noted above, many changes can be made without departing from the spirit and scope of the invention. Accordingly, the scope of the invention is not limited by the disclosure of the preferred embodiments. Indeed, the invention should be defined with full reference to the following claims.

The claims (modification according to treaty clause 19)

1. A method of producing a recombinant polypeptide or protein, comprising transfecting a pluripotent stem cell with a nucleic acid encoding the polypeptide or protein under conditions wherein the polypeptide or protein is expressed by the stem cell, wherein the spPSC is produced from a cell of an animal.

2. The method of claim 1, wherein the polypeptide or protein is selected from the group consisting of: erythropoietin, factor VIII, factor IX, thrombin, antibodies or antibody fragments, interferon alpha 2A and 2B, interferon beta, growth hormone, antihemophilic factor, G-CSF, GM-CSF, soluble receptors, TGF-beta, bone morphogenic protein BMP, TGF alpha, interleukin 2, beta-glucocerebrosidase or an analog thereof, alpha 1-protease inhibitors, fibrin, fibrinogen, von Willebrand factor, imiglucerase, galactosidase beta, Raronidase, glucosidase alpha, thyrotropin alpha, and thymosin alpha.

3. The method of claim 1, wherein the antibody or antibody fragment binds to a target, wherein the target is selected from the group consisting of: tumor necrosis factor TNF molecules, tumor necrosis factor receptor TNFR, growth factor receptor, vascular endothelial growth factor VEGF molecules, interleukin 1, interleukin 4, interleukin 6, interleukin 11, interleukin 12, interferon gamma, nuclear factor kappa-B receptor activator ligand RANKL, and Blys.

4. The method of claim 1, wherein the soluble receptor binds to a target selected from the group consisting of: TNF α, TNF β and Blys.

5. The method of claim 1, further comprising immortalizing the transfected spPSC.

7. The method of claim 5, further comprising inducing differentiation of the transfected immortalized spPSCs.

8. The method of claim 1, further comprising inducing differentiation of the transfected spPSC.

9. The method of claim 7, further comprising immortalizing the transfected differentiated cells.

10. The method of claim 1, wherein the spPSC is selected from the group consisting of: induced pluripotent stem cells, pluripotent stem cells produced by somatic cell nuclear transfer (SCNT pluripotent stem cells), pluripotent stem cells produced by reprogramming assisted by altered nuclear transfer oocytes (ANT-OAR pluripotent stem cells), and pluripotent stem cells produced by parthenogenesis (PGA pluripotent stem cells).

Claims (9)

1. A method of producing a recombinant polypeptide or protein, comprising transfecting a pluripotent stem cell spPSC or endogenous pluripotent stem cell ePSC, produced synthetically by the polypeptide or protein, with a nucleic acid encoding the polypeptide or protein under conditions in which the polypeptide or protein is expressed, wherein the spPSC or ePSC is produced or isolated from a cell of an animal.

2. The method of claim 1, wherein the polypeptide or protein is selected from the group consisting of: erythropoietin, factor VIII, factor IX, thrombin, antibodies or antibody fragments, interferon alpha 2A and 2B, interferon beta, growth hormone, antihemophilic factor, G-CSF, GM-CSF, soluble receptors, TGF-beta, bone morphogenic protein BMP, TGF alpha, interleukin 2, beta-glucocerebrosidase or an analog thereof, alpha 1-protease inhibitors, fibrin, fibrinogen, von Willebrand factor, imiglucerase, galactosidase beta, Raronidase, glucosidase alpha, thyrotropin alpha, and thymosin alpha.

3. The method of claim 1, wherein the antibody or antibody fragment binds to a target, wherein the target is selected from the group consisting of: tumor necrosis factor TNF molecules, tumor necrosis factor receptor TNFR, growth factor receptor, vascular endothelial growth factor VEGF molecules, interleukin 1, interleukin 4, interleukin 6, interleukin 11, interleukin 12, interferon gamma, nuclear factor kappa-B receptor activator ligand RANKL, and Blys.

4. The method of claim 1, wherein the soluble receptor binds to a target selected from the group consisting of: TNF α, TNF β and Blys.

5. The method of claim 1, further comprising immortalizing the transfected spPSC.

7. The method of claim 5, further comprising inducing differentiation of the transfected immortalized spPSCs.

8. The method of claim 1, further comprising inducing differentiation of the transfected spPSC or ePSC.

9. The method of claim 7, further comprising immortalizing the transfected differentiated cells.

10. The method of claim 1, wherein the spPSC is selected from the group consisting of: induced pluripotent stem cells, pluripotent stem cells produced by somatic cell nuclear transfer (SCNT pluripotent stem cells), pluripotent stem cells produced by reprogramming assisted by altered nuclear transfer oocytes (ANT-OAR pluripotent stem cells), and pluripotent stem cells produced by parthenogenesis (PGA pluripotent stem cells).