NZ613888B2 - Personalized production of biologics and method for reprogramming somatic cells - Google Patents

Abstract

Disclosed is a method for producing a therapeutic recombinant biologic polypeptide or protein for treating a disease comprising transfecting a synthetically-produced Pluripotent Stem Cell (spPSC) with a nucleic acid that encodes for the therapeutic recombinant biologic polypeptide or protein, under conditions wherein the polypeptide or protein is expressed by the spPSC, wherein the spPSC is produced from a cell of an animal that is not within a human body. Also disclosed is the use of the therapeutic recombinant biologic polypeptide or protein for treating a disease. conditions wherein the polypeptide or protein is expressed by the spPSC, wherein the spPSC is produced from a cell of an animal that is not within a human body. Also disclosed is the use of the therapeutic recombinant biologic polypeptide or protein for treating a disease.

Description

PERSONALIZED PRODUCTION OF BIOLOGICS AND METHOD ROGRAMMING SOMATIC CELLS THERESA M. R CROSS-REFERENCE TO RELATED APPLICATIONS This application claims ty to US ional Patent Application Serial No. 61/429,409 filed January 3, 2011; 'US Provisional Patent Application Serial No. 61/431,376 filed January 10, 201 1. All of the foregoing applications are hereby incorporated by reference in their entirety.

BACKGROUND OF THE INVENTION There are presently many recombinant polypeptides and proteins used therapeutically to treat a number of diseases. These recombinant polypeptides and proteins are all commercially manufactured using either primary, continuous man, or human diploid cell lines. For instance, some are manufactured using bacteria such as E. Coli, while others are manufactured using yeast or various ovarian ceil lines of animal origin. Bacteria cannot be used for the manufacture of some polypeptides and proteins particularly when glycosylation patterns and other n modifications are critical for biologic-receptor binding affinity, biologic activity, biodistribution or phannacokinetics of the biologic or for recipient immune recognition. When glycosylation is a critical variable, the Chinese Hamster Ovary Cell is currently one of the most commonly used cell lines for biologic manufacture.

Unfortunately, the need to use a recombinant polypeptide or protein for long periods of time or for chronic therapy can result in the patient developing neutralizing, antibodies to the product, making the patient less responsive or unresponsive to the drug. In some cases the patient can switch to another drug in the same class, such as with the variety of anti-TN‘F biologics, such as Enbrel,‘also known as Etanercept; Remicaide, also known as lnfliximab; Certolizumab, and Humira, also known as D257, that are used to treat diseases such as rheumatoid arthritis, juvenile rheumatoid arthritis, sis, tic arthritis, sing spondolytis, ulcerative colitis and s disease routinely. However, generation of neutralizing antibodies to one particular NF biologic in general predisposes the patient to ultimately generate neutraiizing antibodies to another anti-TNF biologic product. In some ces, there are no alternative treatments for the patient, so this neutralizing dy ion leaves the patient without treatment options. Even when there are appropriate treatment options, the pre-disposition to developing neutralizing antibodies to other drugs in the same class ultimately means that these patients may be left without treatment s. [00041 Other biologic products that can be neutralized by human antibodies include: Natalizumab, or Tysabri, an innovative therapy for multiple sclerosis, and Denosumab, a fully human monoclonal antibody to receptor activator of nuclear factor kappa-B ligand (RANKL), approved for the ent of osteoporosis and chemotherapy'induced bone fracture, with potential use to treat breast cancer caused by HRT and hormonal contraceptives. Abatecept is a CTLA-4 fusion n approved for use in rheumatoid arthritis patients who have become refractory to anti-TNF therapies. Although its use is too new to have evidence about Abatecept induced neutralizing antibodies, it also has the possibility that it may induce a human antibody neutralizing antibody reaction.

Other polypeptides and proteins besides the antibody and fusion protein based drugs have also been shown to elicit immune responses with long-term treatment. For instance, recombinant human erythropoietin elicits lizing antibody formation that reduces its efficacy and can lead to a rare a me. Also, the generation of antibodies to coagulation factor therapy for hemophiliacs is as high as 25-30% of the patients.

This is a general problem with the coagulation factors. inant interferon alpha 2a therapy for cancer and hepatitis B also are hampered by the generation of neutralizing dies to the treatment. Refractoriness to tong-term growth hormone therapy for children with short stature is also problematic. There are reports of neutralizing dy formation to some insulin products.

Other ic drugs that have the potential to elicit neutralizing antibodies include whole blood, serum, plasma pools or other y sources of biologic supply, for instance, human albumin, human Alpha 1-Proteinase Inhibitor, human Antihemophilic Factor/von Willebrand Factor Complex, BabyBig Botulism Immune Globulin Intravenous, C1 Esterase Inhibitor, fibrin sealant, fibrinogen, Immune Globulin Intravenous, Immune Globulin Subcutaneous, Protein C Concentrate, Rho(D) Immune Globulin Intravenous, thrombin, von Willebrand Factor/Coagulation Factor VIII Complex.

Recombinant polypeptides and proteins elicit immune ses and neutralizing antibody generation based on multiple characteristics, including: the time frame of biologic ent, the interval of repeat therapy, the amino acid composition of the biologic, and the modifications to the biologic such as glycosylation, methylation, nitrosylation, sialylation, phosphorylation, ion, prenylation, selenation, tinylation, vitamin-dependent modifications, n binding associations, acylation, glycation, 3 dimensional configurations and oiling. Thus there is a need for methods of producing polypeptide protein products having reduced levels of antigenicity in an animal being treated with a biologic product.

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

DESCRIPTION OF THE INVENTION [0008a] In a first aspect, the present invention provides a method for producing a therapeutic recombinant biologic polypeptide or protein for treating a disease comprising transfecting a synthetically-produced Pluripotent Stem Cell (spPSC) with a nucleic acid that s for said therapeutic recombinant biologic polypeptide or protein, under ions wherein the polypeptide or protein is expressed by said spPSC, wherein the spPSC is produced from a cell of an animal that is not within a human body. [0008b] In a second aspect, the t invention provides use of a eutic recombinant biologic polypeptide or protein obtained from a synthetically-produced Pluripotent Stem Cell (spPSC) d from the species of an animal suffering from a disease, in the preparation of a medicament for treating said disease in the animal, - 3a - wherein the spPSC has been transfected with a nucleic acid that encodes for the therapeutic recombinant biologic polypeptide or protein. [0008c] In a third aspect, the t invention provides a therapeutic recombinant biologic polypeptide or protein for treating a disease produced by the method of the first aspect.

The present invention fills this need by providing for methods to produce ics such as polypeptides or proteins, nucleic acids, s and vaccines by transfecting or transforming synthetically produced pluripotent stem cells (spPSCs) or endogenous otent stem cells (ePSC). These cells are derived from the species that is being treated and are transfected with vectors that express the desired biologic and induce expression of the biologic t by the transfected or transformed spPSC or ePSC.

The present invention further provides for a method for producing a recombinant polypeptide or protein comprising producing spPSCs from adult cells or ing ePSCs of an animal and transfecting or transforming said spPSCs or ePSCs with a nucleic acid that encodes for said ptide or protein under conditions wherein the polypeptide or protein is expressed by said stem cell.

In an alternative embodiment of the present invention the spPSCs are produced or ePSCs are isolated from celts of the same ethnic group as that of the individual who is being administered-the recombinant polypeptide or n. Different ethnic groups may have varying glycosylation patterns and distinct polymorphisms from one group to another. Ethnic groups are those groups that have the same blood- or tissue-types. Thus, according to the present invention recombinant ptides and proteins are ed from sp‘PSCsior ePS‘Cs wherein the ePSCs are or spPSCs are produced from cellsisolated from the same ethnic group as the individual being administered the polypeptide or protein. An individual will be administered a recombinant polypeptide or protein produced from an spPSC or ePSC wherein the spPSC is manufactured or the ePSC is isolated from a cell belonging to the ethnic group to which the individual belongs.

The present invention further provides for a method of administering a polypeptide or protein to an individual animal comprising producing spPSCs such as induced pluripotent stem cells (i'PSCs) from said celis of said animal or isolating ePSCs and transfecting or transforming said spPSCs or ePSCs with a nucleic acid that encodes for said ptide or n under conditions wherein the polypeptide or protein is sed by said pluripotent stem cell, isolating said polypeptide or protein from said induced pluripotent stem cell and administering said isolated polypeptide or protein to said individual.

The present invention provides a method for personalized tion of polypeptide or protein therapeutics y making spPSCs or ePSCs commercially viable and useful. The t invention relates to methods on how to make patient ic spPSCs or ePSCs to manufacture patient specific polypeptides or'proteins to overcome issues of neutralizing antibody ion that occur commonly with ptides or n for long term or chronic use. The patient can be any animal, preferably ian and more preferably human. The present invention also provides a method for producing nucleic acids or vimses comprised of transfecting or transforming spPSCs or ePSCs with'a vector under conditions wherein the desired nucleic acid or virus is produced.

Furthermore, the present,invention provides methods to derive a patient- specific and organ or cell-type specific cell line for the production of closely matched post. translationally modified biologics for therapeutic use. Patient specific stem cells can be derived using SCNT, induced reprogramming, parthenogenesis, or ANT-OAR reprogramming methods, or they can be isolated from the target patient. otent stem cells thus derived or isolated can be genetically modified using standard molecular biotechnology procedures to express the therapeutic of interest, for instance using insertional or episomal expression vectors or homologous recombination methods. The genetically modified cell line can be expanded in culture, and banked for ic biologic production runs that would be scheduled based on the shelf-life of the produced biologic (EXAMPLE 2).

Alternatively, the patient c stem cell lines derived can be differentiated in culture towards a cell type that nonnally expresses the highest levels of the desired therapeutic protein and then used for biologic manufacture. Differentiation can be d out for each production run, or could be done on large scale and the differentiated patient specific cell lines banked for subsequent production runs based on the shelf life of the therapeutic produced (EXAMPLE 3).

Additionally, since glycosylation patterns and other post-translational modifications are known to differ among s and cell types, patient c stem cell lines can be prepared from adult or somatic cells isolated from the organ. or from among the cell-type that endogenously expresses the biologic. As an example, SCNT, PGA, ANT-OAR or rammingtechniques can then be applied to derive a pluripotent cell line for biologic production. Pluripotent stem cells thus derived or isolated can be genetically modified using standard molecular biotechnology procedures to express the eutic of interest. The genetically modified cell line can be expanded in culture, and banked for periodic biologic production runs, that would be scheduled based on the shelf-life of the produced biologic (See Example 4). Taking flirther 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 towards their cell-type of origin to more fully create a cell line capable of endogenous post-translational modification. The iPS cells may be genetically modified to s the therapeutic of st prior to re-differentiation towards the original isolated cell- type or afler re-differentiation towards the original cell type (See Example 5).

AFor instance, growth homtone is ly most highly produced by somatotrophic cells in the anterior pituitary, and is also highly sed in cells within the placenta (trophoblasts) and the tongue and vulva or anal skin. Factor VIII protein sion is high in tubule cells in the kidneys, and is moderately expressed by multiple s and cell types, accmding to the Human Protein Atlas. Antibodies are typically produced by B cells which mature in germinal centers of the spleen and other lymphoid organs. Therapeutic production of antibodies with high'levels of antibody-dependent cell-mediated cytoxicity (ADCC) are determined by the level of GDP-D-mannose-4,6-dehydratase (GMD) that can place N-acetylglucosamine (GlcNac) at the bisecting position of lgGlsubtype antibodies present in the manufacturing cell line (.1 Bio! Chem. 1998 and , Vol. 273, pp. l4582—l4587, BMC Bio/ec/mol., 7 (2007). Production of dies with high ADCC activity is not always optimal using the CHO manufacturing cell line ( J Biol Chem. 278: 3466-3473, 2003). .The present invention provides novel ian cell lines for optimal ADCC activity of manufactured antibodies, fusion proteins and cell cytotoxic biologics.

Transcription factors known to be ated with high levels of biologic production can be co-transt‘ected with the gene of interest to optimize expression levels from the patient ic cell line. For instance, high levels of Pit-l expression may lead to high prolactin expression in a cell type while blocking or preventing growth hormone expression (Genes Dev, 3: 946-958 1989).

Monoclonal antibody production can be enhanced by optimizing the gene codon using systems such as those ped by Sino Biological Inc, Peoples ic of China,’ morphogenics (Proc Nall Acad Sci, [03: 3557-3562, 2006), or other standard biotechnology methods.

Production of Synthetically Produced-Pluripotent Stem Cells Any type of synthetically produced otent stem cell can be used to produce the personalized biologics of the present invention. The two main categories are .induced or reprogrammed Pluripotent Stem. Cells (iPSCs) and stem cells produced by nuclear transfer , ANT-OAR and parthenogen'esis.

Somatic cell nuclear transfer (SCNT) isa technique wherein an enucleated egg is injected with the nucleus of an adult somatic cell and implanted in a prepared recipient uterus with resulting live births that yield complete nuclear genetic . Additionally, pluripotent stem cells have been d in culture from SCNT methods (Cell ram. 122l05-l 13, 2010 and Genome R651, [9: 2193-220l, 2009).

Altered nuclear transfer oocyte assisted reprogramming (ANT-OAR) is a technique similar to SCNT, however, the donor nucleus is genetically altered prior to injection into the recipient egg such that the ANT-oocyte is prevented from differentiating and forming a complete organism (Genome Res. 19: 2193-220] , 2009). [0022'] Panhenogenesis (PGA) is also used to generate pluripotent stem cells, using techniques such as zona-free r er, parthenogenetic activation; and cloning techniques such as SCNT and parthenogenesis (PGA) have also been used to generate reprogrammed pluripotent stem cells (CeII Reprogram. 12: , l05-1l3, 2010 and Nalure, 450:497v502 2007).

These pluripotent stem cells can be maintained for somewhat long temi indefinite periods in culture, making them a potential source for biologic manufacturing such as for recombinant proteins, DNA, and viruses.

Induced or Reprogrammed spPSC Induced Pluripotent Stem Cells are similar to natural pluripotent stem cells, such as embryonic stem (ES) cells, in many respects, such as the expression of certain stem cell genes and proteins, chromatin ation patterns, doubling time, embryoid body formation, ma formation, viable chimera formation, and y and differentiability.

,Induced pluripotent stem cells, commonly abbreviated as iPS cells or iPSCs are a type of pluripotent stem cell artificially d from a non-pluripotent cell, typically an adult somatic cell, by inducing a "forced" expression of specific genes (Nature Reports Stem Cells. 2007).

Induced Pluripotent Stem Cells (iPSCs) are produced by transfection of certain stem~ce|l associated genes into non-pluripotent cells. Induced pluripotent stem cells are typically derived by transfection of certain stem cell-associated genes into non-pluripotent cells such as adult fibroblasts. Transfection is typically achieved through viral vectors, such as iruses or retrotransposons. Transfected genes include the master transcriptional tors 4 (Poqul) and Sox2, although it is suggested that other genes enhance the efficiency of ion. After 3-4 weeks, small numbers of transfected cells begin to become morphologically and biochemically similar to pluripotent stem cells, and are typically isolated through morphological selection, doubling time, or through a reporter gene and antibiotic selection. nic stem cell d fibroblasts and adult fibroblasts and other cells have been reprogrammed to a pluripotent state by fusion with embryonic stem cells (Cell. [26:652-655, 2006 and Stem Cell Rev, 2: 0, 2006), by the addition of 4 genes using retroviral transfection ques ( Cell. [26:663-676, 2006), by a single cassette or bicistronic lentiviraltransfection methods [Stem Cells, 27:543-549, 2009, and .S'Iem Cells. 27:1042-1049, 2009), and by endogenous stimulation of pluripotency required transcription factors (Stem Cells. , 27:3053-3062, 2009). ion of pluripotency could also be achieved ‘ by modifying methylation or polyadenylation status of the genome (PLoS One, 4ze84l9, 2009), by microRNAs (Dev Biol, 34446-25, 2010), small le activators of required transcription factors, epigenetic reprogramming (Regen Med, 2:795-816, 2007), by protein- based reprogramming, (Blood ll 6: 386-395, 2010) by addition of culture supernatant or cell ts from pluripotent cells in culture, by chemical or radiation or other means of genetic mutation to reactivate pluripotency genes, and by addition of growth factors or cytokines or cellular signaling moieties that induce or maintain endogenous pluripotent states. [0028| The use of iruses to re-program cells to pluripotent states ts dangers that recall the gene therapy trials for immune deficiency. Excision techniques such as Cre-lox have been used to eliminate the retrovirus after successful reprogramming and piggyBac transposon methods totally eliminate the need for retroviruses (Curr Opin Bi()Iec/mol., 20:5 l 6-52 I, 2009). [0029l Human iPSCs have been produced by transforming human fibroblasts into pluripotent stem cells using four pivotal genes: Oct3/4, 80x2, Klf4, and c-Myc with a retroviral system. Human iPSCs have also been produced using OCT-4, SOXZ, NANOG, and a different gene LIN28 using a lentiviral system. Adenovirus has also been used to ort the requisite four genes into the DNA of skin and liver cells of mice, resulting in cells identical to embryonic stem cells (Science 03):945-949, 2008). Reprogramming of adult cell to iPSCs can also be accomplished via plasmid t any virus transfection system at all (Science 322(5903):949-953, 2008). iPSCs have been produced using the piggy bac oson system, mini circle technology, protein stimulated or ioned media stimulated reprogramming.

The generation of iPS cells is crucially dependent on the genes used for the induction. Oct-3/4 and certain members of the Sox gene family (Soxl, 50x2, 80x3, and 80x15) have been identified as crucial transcriptional regulators involved in the induction process whose absence makes induction impossible. Additional genes, however, including certain members of the Klf family (Klfl, Kle, Klf4, and KlfS), the Myc family (C—myc, L- myc, and N-myc), Nanog, and LIN28, have been identified to increase the ion ncy. o Oct-3/4 (Poqul) (cDNA available from ne, San Diego CA) ( c Acids Res. 20 (17): 4613—20, 1992): Oct-3/4 is one of the family of octamer ("Oct") transcription factors, and plays a crucial role in maintaining otency. The absence of Oct-3M in Oct-3/4+ cells, su‘ch as biastomeres and embryonic stem cells, leads to spontaneous trophoblast differentiation, and presence of Oct-3/4 thus gives rise to the pluripotency and differentiation potential of nic stem cells. Various other genes in the "Oct" family, including Oct-314's close relatives, Octl and Oct6, fail to elicit induction, thus demonstrating the exclusiveness of Oct-3/4 to the induction process.

Sox family: The Sox family of genes is associated with maintaining pluripotency similar to Oct-3/4, although it is associated with otent and unipotent stem cells in contrast with Oct-3/4, which is exclusively exp’ressedin pluripotent stem cells (Dev Biol. 227 ('2): 239—55, 2000).While Sox2 (cDNA available from Bioclone, San Diego, CA) was the l gene used for induction (Mamm. Genome 5 (10): 640-642, 1995), other genes in the Sox family have been found to work as well in the induction process. Soxl(cDNA ble from ne, lnc., San Diego, CA) yields iPS cells with a similar efficiency as 80x2, and genes 50x3 (human cDNA available from Bioclone, Inc., San Diego, CA), SoxlS, and 80x18 also generate iPS cells, although with decreased efficiency.

Klf family: Klf4 of the Klf family of genes is a factor for the generation of mouse iPS cells. Klf). (cDNA available from Bioclone, Inc, San Diego, CA) and Klf4 (cDNA available from Bioclone, Inc, San Diego, CA) are factors capable of ting iPS cells, and related genes Klf I (cDNA ble from Bioclone, Inc., San Diego, CA) and Klf5(cDNA available from Bioclone, lnc., San Diego, CA) did as well, although with reduced efficiency.

Myc family: The Myc family of genes are prom—oncogenes implicated in cancer. C- myc (cDNA available from Bioclone, Inc., San Diego, CA) is a factor implicated in the generation of mouse iPS cells. However, c-myc may be unnecessary for generation of human iPS cells. Usage of the "myc" family ofgenes in induction of iPS cells is troubling for the ality of iPS cells as clinical therapies, as 25% of mice transplanted with c-myc-induced iPS cells developed lethal leratomas. N-myc (cDNA available from Bioclone, Inc., San Diego, CA) and L-myc have been identified to induce instead of c-myc with similar efficiency, Nanog: (cDNA ble from Bioclone, Inc, San Diego, CA) In embryonic stem cells, Nanog, along with Oct-3/4 and 80x2, is necessary in promoting pluripotency (Cell 113 (5): 643—55, 2003).

LIN28: (cDNA available from Bioclone, Inc., San Diego, CA) LIN28 is an mRNA binding protein sed in embryonic stem cells and embryonic carcinoma cells associated with differentiation and eration (Dev Biol 258 (2): 432—42, 2003).

Identity of Synthetically Produced-Pluripotent Stem Cells The generated spPSCs are remarkably similar to lly isolated pluripotent stem cells (such as mouse and human embryonic stem cells (ESCs), mESCs and hESCs, respectively) in the following respects, thus confirming the ty, authenticity, and pluripotency of spPSCs to naturally isolated pluripotent stem cells: Cellular biological properties: 0 Morphology: iPSCs are morphologically similar to ESCs. Each cell has round shape, large nucleolus and scant cytoplasm. Colonies of iPSCs are also similar to that of ESCs. Human iPSCs form sharp-edged, flat, tightly-packed colonies similar to hESCs and mouse iPSCs form the colonies similar to mESCs, less flat and more aggregated colonies than that of hESCs.

Growth properties: Doubling time and c activity are cornerstones of ESCS, as stem cells must self-renew as part of their definition. iPSCs are mitotically active, actively self-renewing, erating, and dividing at a rate equal to ESCs.

Stem cell markers: iPSCs express the same cell surface antigenic markers expressed on ESCs. Human iPSCs express the markers specific to hESC, including SSEA-3, SSEA—4, TRA-l-GO, TRA—l—Sl, TRA49/6E, and Nanog. Mouse iPSCs expressed SSEA-l but not SSEA-3 nor SSEA-4, similarly to mESCs.

Stem Cell Genes: iPSCs express genes expressed in undifferentiated ESCs, including Oct-3/4, 80x2, Nanog, GDF3, REXl, FGF4, ESGl, DPPAZ, DPPA4, and hTERT.

Telomerase activity: Telomerases are necessary to sustain cell division unrestricted by the Hayflick limit of ~50 cell divisions. hESCs express high telomerase activity to sustain self-renewal and proliferation, and iPSCs also demonstrate high telomerase activity and s hTERT (human telomerase reverse transcriptase), a ary component in the telomerase protein complex. otency: iPSCs are capable of differentiation in a n similar to ESCs into fully differentiated tissues: 0 Neural differentiation: iPSCs can entiate into neurons, expressing Blll-tubulin, tyrosine ylase, AADC, DAT, ChAT, LMXlB, and MAP2. The ce of catecholamine-associated enzymes may indicate that iPSCs, like hESCs, may be differentiable into dopaminergic neurons. Stem cell-associated genes are downregulated after differentiation.

Cardiac differentiation: iPSCs can differentiate into cardiomyocytes that ' spontaneously begin beating. Cardiomyocytes expressed TnTc, MEFZC, MYL2A, MYHCB, and NKX2.5. Stem cell-associated genes were downregulated after differentiation.

Teratoma formation: iPSCs injected into immunodeficient mice spontaneously fonn mas after nine weeks. Teratomas are tumors of le lineages containing tissue derived from the three germ layers nn, rm and ectodenn; this is unlike other tumors, which typically are of only one cell type. Teratoma ion is a landmark test for pluripotency.

Embiyoid body: hESCs in culture spontaneously form ball-like embryo-like structures termed “embryoid bodies”, which consist of a core of mitotically active and differentiating hESCs and a periphery of fully differentiated cells from all three germ layers. iPSCs also form embryoid bodies and have peripheral entiated cells.

Tetraploid complementation: iPS cells from mouse fetal fibroblasts injected into loid blastocysts (which themselves can only form extra-embryonic tissues) can form whole, non-chimeric, fertile mice, although with low success rate. The tetraploid complementation assay is a technique in biology 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 ons on embryonal development, and in the study of pluripotent stem cells.

Induced pluripotent stem cells (iPS) have been generated from gut mesentery cells (Cell Reprogram, -247, 2010), retinal pigmented epithelial cells (Stem Cal/3., 28:1981-1991, 2010), amnion cells rentiation, 80:123-129, 2010), fibroblasts ( J Vis Exp. adult neural cells (Nature. , , 2009), Vol. 454, pp. 646-650, 2008), dental pulp (J Dem .Res, Vol. 89, pp. 773-778,2010), adipose Cells (Cell Tran.\'p/anl., -536, 2010), ovarian (J Reprod Dev, 56:481-494, 2010), and many other cells from embryonic, fetal and adult sources. iPS cells can be produced from any cell type, theoretically, although all 220 cell types of the body have not yet been systematically investigated. Several recent studies have demonstrated that iPS cells retain a ‘memory’ for their cell type of origin. This -1}- translates to a preference of iPS cells to re-differentiate in culture, sometimes spontaneously, most readily towards their cell type of .

Isolation of Endogenous Stem Cells Stem cells, including endogenous pluripotent 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 arid Nanog and other markers, among other methods. The y type of endogenous pluripotent stem cell that has been isolated to date is the very small embryonic-like (VSEL) stem cells.

VSELs are small (3-5 micron diameter in the mouse and 3-7 micron diameter for , with high nuclear to cytoplasmic ratio. ,VSELS are positive for SSEAl, Oct4, Nanog, Rex'l and other pluripotent stem cell markers, and for CDl33, CD34, AP, cMet, LIF- R, and CXCR4, ( J Am Coll Cardiol l0-20, 2009; Stem Cell Rev 4:89—99, 2008).

They are negative for CD45. VSELs are smaller than HSCs (3—6 vs. 6-8 pm) and have higher nucleus/cytoplasm ratio. The VSEL nucleus is large, contains open-type‘ tin and is surrounded by the narrow rim of cytoplasm with us mitochondria. Therefore, their logy is consistent with primitive PSC.

The absolute numbers of circulating VSELs invPB are exceptionally low (I to 2 cells in 1 mL of blood under steady—state conditions) and thus special flow cytometric protocols have to be applied for their identification and isolation. Phenotypic markers used to identify VSELs include negative expression of CD45 (mouse and human), positive expression of Sca-l (mouse), CXCR4, CD133 and CD34 (mouse and , positive for progenitor stem cell markers (that is, Oct-4, Nanog and SSEA) and express several s characteristic of epiblast/germ line stem ceils.

Employing only fluorescence activated cell sorter isolation of VSELs, sorting of all the VSELs present in 100 mL of UCB can be complete within 4 working days. A more efficient and economic three-step isolation protocol allows recovery of 60% of the initial number of Lin—ICD45—lCDl33b UCB~ VSELs. The protocoi includes lysis of erythrocytes in a hypotonic um chloride solution, CD133b cell selection by immunomagnetic beads and sorting of Lin—/CD45—/CDI33p cells by FACS with size-marker bead controls.

The isolated cells are highly enriched for an Oct-4b and SSEA-4b population of small, highly primitive Lin-/CD4S—/CD 1 33b cells.

Another method for sorting of VSELs is based on the presence of l surface markers and the diameter of the cells. Briefly, the initial step is the lysis of red blood cells to obtain the fraction of nucleated cells. Erythrocyte lysis buffer is used instead of Ficoll centrifugation because the latter approach might deplete the population of very small cells.

Subsequently, cells are stained with antibodies against Sca-l (murine VSE'Ls) or CD133 (human VSELS), panhematopoietic antigen (CD45), hematopoietic lineages markers (lin), and CXCR4 and sorted using a multiparameter, live sterile cell g systems (Mo'Flo, Beckman Coulter; FACSAria, n Dickinson). This method uses “extended lymphocyte gate" to include events with diameter 2—10 pm, including imately 95% of VSELS.

Endogenous stem cells may be contained within the mononuclear cell fraction from bone marrow, whole blood, umbilical cord blood, adipose tissue or other sources, or they may be purified by selection for CD34, CD133, CDIOS, CD1 17, SSEAl-4, dye - exclusion or other specific stem cell antigens. Stem cells can be isolated from whole blood, bone marrow, umbilical cord blood, adipose tissue, tissue scrapings from the olfactory mucosa and other stem cell sources that can be dissociated into single cell suspensions, such as umbilical cord tissue, by density gradient centrifugation using Ficoll-Hypaqueor other commercially available gradients. Stem cells can be recovered from the mononuclear cell fraction resulting from such procedures. Alternatively, stem cells can be found within other ons after density nt centrifugation (Stem Cells Dev. 2011 [Epub ahead of print].) For instance, umbilical cord blood can be diluted 1:1 in PBS, carefully overlaid onto Histopaque 1077 (Sigma) and centrifuged at 1500 rpms at room temperature for 30 minutes.

The resulting layers as depicted can be further processed for stem cell ion. Layer 1 is the et layer, layer 2 is the buffy coat containing mononuclear cells, layer 3 is the Ficoll . layer, and layer 4 is the red blood cell. pellet layer, which also contains VSELs. Layers l, 2, and 3 can be collected, diluted with riate media such as DMEM F12 with or t FBS and centrifuged again to obtain the cell pellet. Layer 4 can be diluted with appropriate media such as DMEM F12 and centrifuged at 800 rpm for 15 minutes at room temperature in a standard benchtop centrifuge. Stem cells can be recovered inantly from layer 2 (Buffy coat) and layer 4 (RBC pellet), following the types of flow cytometric methods ed above. Fig I shows a typical view of layers resulting from gradient fugation of whole blood. I shows the platelets; 2 the bufi‘y coat with MNCs and stem cells; 3 the ficoll; and 4 the RBC pellet and stem cells.

Additionally, since glycosylation patterns and other post-translational ations may differ among tissues and cell types, patient specific stem cell lines can be prepared from adult or somatic cells isolated from the organ or from among the cell-type that endogenously expresses the biologic. See Rajpert-De Meyts 'E, et al. "Changes in the profile of simple mucin-type O-glycans and ptide GalNAc-transferases in human testis and testicular. neoplasms are associated with germ cell maturation and tumour differentiation"., Virchows Arch, Vol. 5-814 (2007) See va M., el al. “Post-translational modifications of tau protein”. Bra/ix/ Lek ljsly,l07:346-353 (2006).

DETAILED DESCRIPTION OF THE RED EMBODIMENT tion of lmmortalized spPSCs and ePSCs in a preferred embodiment of the present invention the spPSCs and ePSCs are immortalized preferably h ral ion of the large T- antigen typically using the polyomavirus simian virus 40 (SV40). See Rose, MR. el al., (I983). "Expression of the Large T Protein of Polyoma Virus Promotes the Establishment in Culture of "Normal" Rodent Fibroblast Cell Lines". PNAS 80: 4354—4358 (1983); and Hofinann, MC. el al. “lmmortalization of germ cells and somatic testicular cells using the SV40 large T antigen” Experimental Cell Research, 201:4]7-435 (1992).

Overview of an Embodiment Using Synthetically Produced, Preferably, Induced Pluripotent Stem Cells or More Preferably Isolated Endogenous Pluripotent Stem Cells In a preferred embodiment: l. Endogenous pluripotcnt stem cells are isolatcd; 2. The ePSC are immortalized; 3. The immortalized ePSC are transfected with the gene, virus or nucleic acids of interest using ral technology; 4. The transfected. immortalized ePSC are then induced to entiate into a germ line cell, preferably an ovary cell, so as to be able to express nucleic acid product in a more efficient manner; . The differentiated cell can now be induced to express the nucleic acid product of interest from the usly transfected vector containing the nucleic acids of interest.

In another preferred embodiment: I. Somatic cells are isolated; 2. The somatic cells are transformed into d pluripotent stem cells (iPS cells); 3.The iPS cells are immortalized; 4. The immortalized iPS cells are transfected with a vector ning the gene, virus or c acids of interest; . The transfected, immortalized iPS cells are then induced to ferentiate into a somatic cell so as to be able to express protein in a more efficient manner; 6. The re-differentiated cell can now be induced to express the protein of interested from the previously transfected vector containing the gene of interest. {0044] In an even more preferred embodiment: l. Endogenous otent stem cells are isolated; 2. The immortal ePSC are transfected with the gene, virus or nucleic acids of interest using non-viral technology; 3. The transfected, immortalized ePSC are then induced to differentiate into a germ line cell, preferably an ovary cell, so as to be able to express nucleic acid product in a more efficient manner; 4. The differentiated cell can now be induced to s the nucleic acid product of interest from the previously transfected vector containing the nucleic acids of st. s to accomplish the red embodiments of this invention are well known to those skilled in the art.

In alternative embodiments the ePSC or spPSC can be transfected with the vector containing the nucleic acids of interest prior to being immortalized. See Du C. er 0/.

“Generation of Variable and Fixed Length siRNA from a novel siRNA Expression ”, Biamed. &. Biophys. Res. Comm. 345:99-105 (2006); York Zhu, US. Patent Application Serial No. 12/313,554 filed on November 21, 2008. Or the ePSC or spPSC cells can be induced to re-differentiate and then the cells can be immortalized and the immortalized, re- differentiated cells can be transfected with a vector containing the nucleic acids of interest.

Another possibility is the ePSC or spPSC celis can be induced to re-differentiate, the re- differentiated cells can be transfected with the nucleic acids of interest and the re- differentiated, transfected cells can be immortalized. it is preferable that the ePSC or spPSC cells be expanded in culture prior to re- entiation, preferably in a cell culture medium containing autologous human serum and stem cell factor or leukemia inhibitory factor.

The polypeptide or protein that is produced can be any polypeptide or protein.

Of particular interest are the polypeptides or proteins selected from the group consisting of erythropoietin, factor VII], factor IX, thrombin, an dylor antibody fragment, alpha interferon, interferon alpha 2A and 23 (See US. Patent Nos. 4,810,645 and 4,874,702), beta interferon (See US Patent No. 4,738,931), consensus interferon (See US. Patent No. ,661,009), growth e, antihemophilic factor, G-CSF, GM-CSF, a soluble receptor, fusion proteins such as the fusion of a soluble receptor to the nt region of an immunoglobulin (lg) (See US Patent No. 5,155,027), TGF-B, bone morphogenic proteins (BMP), TGFa, interleukin 2, ocerebrosidase or an ue f, alphal-proteinase inhibitor, fibrin, fibrinogen, von Willebrand factor, imiglucerase, agalsidase beta, laronidase, alglucosidase alpha, alglucosidase alpha, thyrotropin alpha, and thymosin alpha.

Any antibody or antibody fragment can be produced according to the process of the present invention. Of particular interest are those antibodies or antibody fragments that bind to a target, wherein said target is selected from the group consisting of a tumor necrosis factor (TNF) molecule, a growth factor or, a ar endothelial growth factor (VEGF) molecule, interleukin 1, interleukin 4, interleukin 6, interleukin ll, interleukin 12, gamma interferon, Receptor activator of nuclear factor kappa-B ligand (RANKL) and Blys.

Induction of entiation of Stem Cells To optimize production of the protein of st, the transfected ePSC or spPSC cells should be induced to differentiate into a somatic cell.

In an alternative ment the population of the ePSC or spPSC cells could be expanded and differentiation can be induced and the differentiated cells could then be transfected with the nucleic acid of interest. Stem celis can be induced to differentiate towards somatic cell types in culture by the addition of various growth factors to the culture (Blood, 85:2414-2421, 1995), by altering nutrients in the culture media, by manipulating culture conditions such as oxygen tension, (BA/[C Cell Biol., 11:94, 2010) or temperature, or by culturing the stem cells on various extracellular matrices, among other methods as are known to those in the field of cellular biology and cell differentiation. For instance, retinoic acid, TGF-B, bone morphogenic proteins (BMP), ascorbic acid, and beta-glycerophosphate lead to tion of osteoblasts; thacin, IBMX (3-isobutyl-l—methy1xanthine), ~16- insulin, and triiodothyronine (T3) lead to production of adipocytes; orFGF, BFGF, vitamin D3, TNF-B and retinoic acid lead to production of myocytes (CARDIAC-DERIVED STEM CELLS. (W0/1999/049015) March 1998'). Germ cells have been generated from pluripotent stem cells using yer e, the formation of embryoid bodies (EBs) , co-aggregation with BMP4-producing cells, and the use of testicular or ovarian cell-conditioned medium, or EB formation with recombinant human bone morphogenetic 'proteins (BMPs) (PLoS One. 2009;4(4):e5338). Germ cell marker genes include PR domain containing 1, with ZNF domain , also known as BLIMPI), PR domain containing 14 (PRDMl4), protein arginine methyltransferase 5 (PRMTS), DPPA3, [FITM3, GDF3, c-KIT, chemokine (C-X-C motif) or 4 (CXCR4), NANOSl-3, DAZL, VASA, PlWI family genes (PIWILI and PIWILZ, known as HIWI and HILI in humans, respectively), Mut—L Homologue-l (MU-ll), synaptonemal complex protein 1 (SCPI), and SCP3. The resulting gemi cell lines can be genetically modified to express gene or protein products of st similarly to the use of Chinese Hamster Ovary (CHO) cells and other currently used manufacturing cells lines.

Differentiation strategies to obtain various somatic cell lines from stem cells at various stages . are well known to those in the field of stem cell biology.

Transcription factors known to be ated with high levels of biologic tion can be co-transfected with the gene of interest to ze expression levels from the patient specific cell line. For instance, high levels of Pit-l expression may lead to high prolactin expression in a cell type while blocking or preventing growth hormone expression (Genes Dev, 3:946-958, 1989).

] Monoclonal antibody production can be ed by optimizing the gene codon using systems such as those developed by Sino Biological Inc, morphogenics, or other rd biotechnology methods. '[0054] According to the present invention recombinant polypeptides and proteins are produced in ePSCs and spPSCs wherein the spPSCs and ePSCsare produced or isolated from cells of a specific breed or ethnic group due to the fact that some breeds or ethnic groups of a specific species of animal have different glycosylation patterns in the polypeptides or proteins produced by the c breed or ethnic group. According to the present invention an ethnic group is a group of people whose members identify with each iother, through a common heritage, often consisting of a common ancestry or endogamy (the practice of marrying within a specific group for example Ashkenazi Jews). In general it is a highly biologically self-perpetuating group. Examples of ethnic groups that may have different glycosylation patterns in the polypeptides and proteins are illustrated in Levinson, David (1998), Ethnic Groups Worldwide: A Ready Reference Handbook, Greenwood Publishing Group.

METHOD AND SYSTEM. FOR STEM CELL THERAPY |0055| The present invention also includes a method for promoting stem cell therapy without formation ofteratomas. The present invention s how to advantageously utilize the observed ‘memory’ of reprogrammed somatic cells herein called synthetically produced Pluripotent Stem Cells ) defined above, for greater therapeutic . The memory of spPSCs, which confers a ence to ferentiate towards the cell type of origin prior to reprogramming, provides a means to enhance the safety and therapeutic utility of spPSCs for rative medicine.

General Steps The present invention involves 1. Isolation of somatic celis of interest especially somatic cells that one wishes to regenerate; 2. Transformation of the somatic cells into tically produced Pluripotent Stem Cells (spPSCs), especially induced Pluripotent Stem Cells (iPSCs) as described above; 3. Expansion of the population of spPSCs n the spPSCs maintain the intrinsic etic memory of the somatic cell; 4. Re-differentiation of the spPSCs in culture to re-differentiated somatic cells having the original somatic cell type; and . Administration or delivery of the re-differentiated somatic cells into the area of the body where the cell-type is desired.

Several recent studies have trated that spPSCs cells retain a ‘memory’ for their cell type of origin (Stem Cal/s. 28: 1981-1991, 2010) , (Na/me, 467(7313):285-90, 2010), (Nat Biotechnol , 28, I 848-855, 2010) and 124171 Reprod.,l6:880-885, 2010).

This translates to a preference of spPSCs to reodifferentiate in culture, sometimes spontaneously, most readily towards their cell type of origin. Scientists and clinician ists have focused with a tunnel determination on generating pluripotent stem cells that might be useful for clinical therapy, for drug discovery, for disease modeling or for ty screening (Curr Opin Biolec/mol., 20: 516-521, 2009), missing the therapeutic advantage that the memory aspect of pluripotent stem cells provides for safe human therapy. Indeed, the -]8- scientists who published data regarding the ‘memory’ aspects of reprogrammed pluripotent stem cells have focused on the limitations that this property of spPSCs cells create for therapy, and have missed the icance of this ty for providing therapeutic utility of these cells The memory aspect of spPSCs has been observed from somatic cells of multiple origins. For instance, y fetal retinal epithelial cells were reprogrammed into iPSCs using lentiviral sion of OCT4, SOXZ, LINZS, and Nanog (.S‘Iem Cells. 28: I981- 1991, 2010), and passed the standard tests for pluripotency; they formed teratomas and sed pluripotent stem cell markers. After removal of basic FGF from the growth media, some of the retinal spPSC lines spontaneously re—differentiated back towards a retinal epithelial cell lineage. Approximately 60% of the cells that spontaneously differentiate from human fetal retinal epithelial spPSCs cells are retinal epithelial cells, compared to between 5 and 16% retinal lial cells from spPSCs from human fetal lung fibroblasts, from human foreskin fibroblasts, or human ESC cells. However, one of the 3 spPSCs cells from human fetal retinal epithelial cells failed to differentiate into retinal lial cells at all. Kim et. al.

(Nature, 467(7313):285-90, 2010), reprogrammed bone marrow progenitor cells and dermal fibroblasts from aged mice using retroviral uction of Oct4,Sox2, Klf4 and Myc. All of their resulting stem cells lines demonstrated pluripotency using the ia typically applied to human samples. Subsequent entiation of their reprogrammed pluripotent stem cells demonstrated that hematopoietic sources ferentiated towards hematopoietic lineages more readily than fibroblast sources, and similarly, fibroblast sources re-differentiated towards mesenchymal lineages more readiiy than hematopoietic sources. The authors also showed that this propensity to re-differentiate preferably s their somatic lineage of origin could be overcome partially by differentiation towards the hematopoietic lineage followed by another round of pluripotent reprogramming and then additional differentiation.

For instance, reprogrammed cells derived from neural progenitor cells were differentiated towards hematopoietic es then reprogrammed to pluripotency and showed higher hematopoietic colony formation than neural progenitors that were differentiated towards neural cells, reprogrammed and then differentiated towards poietic cells. {0059] Similar observations of a preference for reprogrammed cells to ferentiate towards their cell type of origin have been made using non-viral reprogramming of human fetal neural progenitor cells (l’LoS One. 4: , e7076-e7088, 2009) . The resulting reprogrammed cells sed several markers of pluripotency, markers for all three germ layers, and were able to form embryoid bodies in culture and teratomas, however, using GeneChip analysis, the authors demonstrated that the rammed neural progenitor cells retained some expression of neural stem cell genes. Polo et.al. (Nat Bimec/mol, 28, : 848-855, 2010), derived pluripotent reprogrammed cells from mouse tail tip derived fibroblasts, splenic B cells, bone marrow granulocytes and skeletal muscle precursors. Autonomous entiation studies indicated that splenic B cell and bone marrow granulocyte reprogrammed spPSCs cells gave rise to hematopoietic progenitors more efficiently than fibroblast or skeletal muscle d spPSCs cells. Interestingly, serial passage of these various spPSCs cell lines led to the abrogation of genetic and methylation differences by passage 16, and the cells also then demonstrated equivalent differentiation efficiencies, in contrast to the earlier results at passage 4. stingly, this phenomenon of differential differentiation potential is not restricted to reprogrammed somatic cells, but has also been observed for nic stem cells lines, which have been found to have differing genetic signatures and spontaneous preference for differentiation towards certain cell lineages (Nat Biotechnol. 26: 313-315, 2008) , (Hum Reprod Update. 13: , 103-120, 2007) (Dev Biol. , 6-459, 2007) (BMC Cell Biol, 10:44, 2009) .

Stem cells can be characterized and isolated by specific antigens expressed on their surface. Pluripotent stem cells canvbe characterized by the expression of stage-specific embryonic antigen (SSEA), the transcription factors Oct4 and Nanog and other markers, among other s. Embryonic stem cell derived asts and adult fibroblasts and other cells have been reprogrammed to a pluripotent state by fusion with embryonic stem cells (Cell 126: 652-655, 2006 and Stem Cell Rev, 2: 331-340, 2006), by the addition of 4 genes using iral transfection techniques (Cell 126: 663-676, 2006) a single cassette , by or bicistronic iral transfection methods (Stem Cells 27: 543-549, 2009 and Stem Cells 27: 1042-1049, 2009), and by endogenous ation of pluripotency required transcription factors (Stem Cells 27: 3053-3062, 2009) . Induction of pluripotency could also be achieved by modifying methylation or polyadenylation status of the genome (l’LoS One 4: e84l9, 2009) , by NAs (Dev Biol. 344: 16-25, 2010), small molecule activators of required transcription factors, etic reprogramming (Regen Med. 2: 795-816, 2007), by n- based reprogramming (Blood 1 16: 386-395, 2010), by addition of culture supematant or cell extracts from pluripotent cells in culture, chemical or ion or other means of genetic mutation to reactivate pluripotency genes, or addition of growth factors or cytokines or - cellular signaling moieties that induce or maintain endogenous pluripotent states. The use of retroviruses to reprogram cells to pluripotent states presents dangers that recall the gene therapy trials for immune deficiency. Excision techniques such as Cre-lox or the piggyBac _20_ transposon methods have been used to eliminate the retrovirus after successful reprogramming (Curr 0pm Bioleclmol. 20: 516-521, 2009). g techniques such as SCNT and parthenogenesis (PGA) (Cell Reprogram. 12: 105-1 13, 2010) have also been used to generate rammed pluripotent stem cells e 450:497—502, 2007).

Significant amounts of resources - economic, intellectual and labor - have been invested in discovering various sources of pluripotent stem cells, primarily in the hopes that these stem cells would be appropriate for human regenerative medicine y.

Unfortunately, except for the adult VSELs (Slam Cell Rev 4:89-99, 2008), all of the pluripotent stem cells isolated to date are thwarted e of problems with teratoma formation, tumor formation, and even neoplastic ties. Therefore, some utility for these pluripotent stem cells is needed in order to take advantage of the economic, intellectual and labor investment that has been made over the past 15 years or so.

Stem cells can be induced to differentiate towards somatic cell types in culture by the addition of various growth s to the culture (Blood 85:2414-2421, 1995) , by ng nutrients in the culture media, by manipulating culture conditions such as oxygen tension (BMC Cell Biol. 11:94, 2010) or temperature, or by culturing the stem cells on various extracellular matrices, among other methods as are known to those in the field of cellular biology and cell differentiation. For instance, retinoic acid, TGF-B, bone morphogenic proteins (BM P), ascorbic acid, and beta-glycerophosphate lead to production of osteoblasts; indomethacin, 'IBMX (3-isobutyl-l-methylxanthine), insulin, and triiodothyronine (T3) lead to production of ytes; aFGF, bFGF, vitamin D3, TNF-B and retinoic acid lead to production of myocytes (W0/1999/0490l5) March 1998) . Differentiation strategies to obtain various c cell lines from stem cells at vari0us stages are well known to those in the field of stem cell biology.

Stem cell therapy is being investigated and perfected for the treatment of many human diseases. Clinical trial information contained in the NIH website www.clinicaltrialsgov lists over 3000 stem cell investigations. Diseases under evaluation include: hematological malignancies, leukemias,‘lymphomas, cancers, osteopetrosis, aplastic anemia and cytopenias, sickle celE e and thalassemia, limbal stem cell deficiency, breast cancer, acute myocardial infarction, coronary artery disease, peripheral vascular disease, heart failure, type '1 diabetes mcllitus, type 2 diabetes us, .strokc, spinal cord injury, neuroblastoma, multiple sclerosis, systemic sclerosis, lupus erythematosus, chronic wound healing, burns, fracture healing, cartilage , CNS tumors, osteoarthritis, renal failure, -2]- Parkinson’s e, myelomas, diabetic foot, liver and biliary sis, dilated cardiomyopathy, anemia, retinitis pigmentosa, Crohn’s e, diabetic neuropathy, mastocytosis, ovarian cancer, epilepsy, myasthenia gravis, autoimmune diseases, granulomatous disease, osteonecrosis, liver failure, PMD disease, lypodystrophy, demyelinating diseases, cartilage s, l disease, lupus nephritis, Alzheimer’s Disease, traumatic brain injury, sarcoma, myositis, hyperglycemia, macular degeneration, ulcerative colitis, muscle degeneration, and others.

Stem cells can be ed using various markers known to those practicing in the field. For instance, a list of common stem cell markers can be found at http://stemcells.nilt.gov/info/scireporti’aggendixe.asp#eii. Neural stem cells can be isolated by the use of CD133; mesenchymal stem and progenitor cells by the use of Bone 'Morphogenic Protein Receptor (BMPR); hematopoietic stem cells by CD34; mesenchymal stem cells by the combination of CD34+Scal+Lin~ markers; hematopoietic and mesenchymal stem cells by ckit, Strol, or Thy}; neural and pancreatic progenitors by nestin; ectoderm, neural and pancreatic progenitors by vimentin; and other markers.

The present invention r es methods to safely reprogram and re- differentiate c cells for rative medicine. Useful somatic cells can include fully differentiated somatic cells, itor cells, or more primitive stem cells. Depending on the organ that requires regenerative therapy, more primitive stem cells may be more or less accessible by organ puncture, biopsy, scrapings or surgical access. Use of more primitive stem cells is preferable when access to these stem cells is possible. Le'ss preferable, but more preferable than fiilly differentiated somatic cells, is the useof‘ progenitor cells.

Isolation of somatic cells from the specific organ ed for treatment, reprogramming of those c cells, short term expansion in culture assuring that the intrinsic ‘memory’ of the spPSCs is maintained, followed by re-differentiation in e to the cell type of origin followed by therapeutic application allows for pluripotent stem cell s to be used to treat patients with reduced dangers of tumor or teratoma formation.

Reprogrammed somatic cells could be used between passages 1 and 12, most preferably at passage 4. The reprogrammed sematic cells could be differentiated towards the cell type for desired regeneration according to standard cell biology and differentiation techniques. The resulting therapeutic cell could be stered by intravenous, intra- arterial, intramuscular or other injection methods using standard injection techniques that may include catheters such as the NOGAStar or MyoStar injection catheters or other approved catheter injection . Alternatively, the therapeutic cells may be administered to the target tissue by minimally ve or more invasive surgical methods. The therapeutic cells may be administered in a buffer composition containing autologous human serum n between 0 and 15%, most preferably 5%. For treating disease of the l nervous system, the therapeutic cells would ably be buffered using autologous cerebrospinal fluid. The therapeutic cells may also be administered through the use of a scaffold such as collagen, fibrinogen or other extracellular matrix or combination of extracellular matrices, or by the use of a thread such as created using alginate or other standard methods, to hold the cells in place and maintain contact with the organ that requires regenerative repair.

Preferably A minimum of 500 therapeutic cells will be administered, and commonly cell therapy will utilize 15 miltion to 500 million cells. More ably, between million and 100 million cells will be administered for optimal .

As an example, for cell therapy for neural diseases such as Alzheimer’s, Parkinson’s, , Huntington’s Disease, le sclerosis, paralysis and iother diseases of the central nervous system (CNS), would preferably isolate olfactory mucosa using rigid endoscopes as published (J Spinal Cord Med. 29:191—203, 2006). Neural stem and itor cells or olfactory ensheathing cells are isolated from the olfactory mucosa according to methods well known to those practicing in the field of regenerative medicine.

Alternatively, neural crest stem cells can be isolated from human hair follicles (Folia Biol . 56:149-157, 2010) .

Most preferably, neural stem cells are rammed by the addition of defined factors, expanded and ed for 4 es, re-differentiated towards specific central nervous system cell types by the addition of defined factors, and then administered to the patient for regenerative therapy.

The invention provides 'methods to safely reprogram and re-differentiate somatic cells for regenerative medicine. Useful somatic cells can include fully differentiated somatic cells, progenitor cells, or more primitive stem cells. Depending on the organ that requires regenerative y, more primitive stem cells may be more or less accessible by organ puncture, biopsy, scrapings or surgical access. Use of more primitive stem cells is preferable when access to these stem cells is possible. Less preferable, but more preferable than fully differentiated sornatic cells, is the use of progenitor cells.

Fer spinal injury , otfactory mucosa is removed by rigid endoscope, neural stem cells are ed using the neurosphere assay first described in 1992 by Reynolds and Weiss (.S‘cience, 255:1707-1710, 1992), epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) are added to the cells that survive several days of culture of the olfactory mucosa to stimulate neurosphere , and neural stem cells are recoverable within 7 to 10 days of culture. The resulting neural stem cells are reprogrammed by the addition of episomal vectors for the delivery of OCT4 and NANOG with hygromycin selection for 5-7 days. The cells are passaged and expanded until passage 4 to obtain integration-free colonies, and then differentiated towards olfactory ensheathing and stem-like neural progenitor cells using autologous cerebrospinal fluid or defined factors. The damaged spinal cord is exposed during surgery using a standard e incision and a posterior midline myelotomy, scar tissue is removed as permissible, the therapeutic cells are buffered in autologous cerebrospinal fluid, seeded onto a bioactive scaffold and applied directly to the injured spinal cord.

EXAMPLES Example 1 Genetic modification of patient specific synthetically ed otent stem cells for inant protein production.

Synthetically ed pluripotent stem cells (spPSCs) such as SCNT or PGA or ANT-OAR or iPSCs are used from patients for genetic modification to induce biologic production. SCNT derived stem cells are prepared by erring the s of a patient’s cell into an enucleated oocyte that has been ed. ANT-OAR derived stem cells are prepared by genetically modifying a patients nuclear DNA prior to transferring the modified nucleus into an ated, ed oocyte. iPSCs derived stem cells are prepared by reprogramming the patient’s cells using genetic modification, activators of pluripotent transcription factors, epigenetic modification or other methods known in the art as described above. The resulting patient c synthetically produced stem cell line is ‘banked’ as a master cell bank and a working bank for subsequent genetic modification for biologic production.

Germ cells are generated from pluripotent stem cells using monolayer culture, the formation of id bodies (EBs), co-aggregation with BMW-producing cells, the use of testicular or ovarian cell-conditioned medium, or E8 formation with recombinant human bone morphogenetic proteins (BMPs). Germ cells are identified by the expression of marker genes that can include PR domain containing 1, with ZNF domain (PRDMl, also known as BLIMPl), PR domain containing 14 (PRDM l4), n arginine methyltransferase 5 (PRMTS), DPPA3, lFlTM3, GDF3, c-KIT, cltemokine (C-X-C motif) receptor 4 (CXCR4), I-3, DAZL, VASA, PlWI family genes ‘(PIWILI and PIWIL2, known as HIWI and HILI in humans, respectively), Mut-L Homologue-1 (MLHI), synaptonemal complex protein 1 (SCPI), and SCP3. The resulting germ cells are transfected with a gene of interest, such as Factor VIII, according to s generally used for the manufacture of recombinant Factor VIII.

Example 2 Genetic modification of patient c synthetically produced pluripotent stem cells for recombinant insulin production.

Synthetically produced pluripotent stem cells (spPSCs) such as SCNT or PGA or ANT-OAR or iPSCs derived stem celis are used from patients for genetic modification to induce biologic production. SCNT derived stem ceils are prepared by transferring the nucleus of a patient’s cell into an enucleated oocyte that has been prepared. ANT-OAR d stem cells are prepared by genetically modifying a patients nuciear DNA prior to transferring the modified nucleus into an enucleated, prepared oocyte. iPSCs derived stem cells are prepared by reprogramming the patient’s cells using genetic ation, activators of otent transcription s, etic modification or other methods known in the art as described above. The ing patient specific synthetically ed stem cell line is ‘banked’ as a master cell bank and a working bank for subsequent genetic modification for ic production.

Expression of insulin precursors in patient specific stem cells is performed according to methods generally used for the manufacture of insulin in S. cerevisiae : [Kjeldsen T., et al., “Engineering-enhanced protein secretory expression in yeast with application to insulin”. 21, May 2002, J Biol Chem, 277:!8245-18248 (May 2002); Zhang B., e! a/., “Intracellular ion of newly sized insulin in yeast is caused by endoproteolytic processing in the Golgi complex”., J Cell Bio/., 153:! 187-1 198 (June2001); and Kristensen C. et a/., “Alanine scanning mutagenesis of insulin”., .1 Biol Chem, 272:12978-12983 (May 1997)], or E. Coii [Son ‘11., e! al. “Effects of beta-mercaptoethanol and hydrogen peroxide on tic conversion of human proinsulin to insulin”., J.

Microbiol Biolechnol.. 18:983-989 (May 2008)], then processed and purified according to standard methods. The insulin precursor expressing patient specific cell line is ‘banked' as a master cell bank and a working cell bank for subsequent insulin production.

Example 3 Generation of ells for insulin production using genetic modification of patient specific synthetically produced pluripotent stem cells.

To produce insulin, somatic cells are used from patients for genetic modification to produce spPSCs and these are used to induce ic production. The spPSCs derived stem cells are prepared by reprogramming the patient’s cells using genetic modification, activators of pluripotent transcription factors, epigenetic modification or other methods known in the art. The resulting patient specific stem cell line is ‘banked’ as a master cell bank and a working bank for subsequent genetic ation for biologic production. Or endogenous pluripotent stem cells (ePSCs) can be isolated according to techniques described above and banked.

Expression of n precursors in patient c stem cells is med according to methods generally used for the manufacture of insulin in S. cerevisiae or E. coli as described above. Following gene transfection with the appmpriate insulin gene constructs, the cells are differentiated towards the beta-cell lineage following standard protocols found in [Shi, Y., e1. All “Inducing nic stem cells to differentiate into pancreatic beta cells by a novel three-step approach with activin A. and all-trans retinoic acid Stem Cal/3., 23:656—662 (2005); or hi, K., er. AI. “Generation of insulin-secreting islet-like clusters from human skin fibroblasts”., J Biol Che/22., 283:31601—31607 (2008)] of the Beta Cell Biology Consortium, http://www.protocolonline.org/prot/Cell_Biology/Stem_Cells/Dif‘ferentiation_0f_Stem_Cell/i ndex.html. Protocol Oil/inc. [Online] [Citedz Dec 19, 2010.] The resulting expressed biologic product is then processed and purified according to standard methods. The resulting patient c insulin precursor expressing stem cell line is ‘banked’ as a master cell bank and a g bank for subsequent c modification for biologic tion.

Altematively, the resulting t specific stem cells are differentiated towards the beta~cell lineage following standard protocols found in Shi er. Al. id. Or Tateishi et. Al. id of the Beta Cell Biology Consortium id. Once differentiated, expression of insulin precursors in patient specific stem cells is med according to methods lly used for the manufacture of insulin in S. cerevisiae : or E. Coli as discussed above. The resulting expressed biologic product is then processed and purified ing to standard methods.

Example 4 Isolation of adult (somatic) antibody producing cells for reprogramming and transfection to produce biologic antibody therapeutics. [0079l dy producing B cells are isolated from peripheral blood, bone marrow and other readily accessible hematopoietic cell sources for the purpose of generating patient c manufacturing cell lines to e therapeutic antibody biologics. B cells are isolated using available kits based on CDI9 expression (StemCell Technologies). Limiting dilution or cell sorting methods may be employed to select cells producing the highest levels of immunoglobulin (lg), Afler brief expansion, the clonal high lg producing cell is reprogrammed to a pluripotent or progenitor state using standard reprogramming techniques.

The ing patient specific stem cells are transduced with the desired antibody gene constructs using standard molecular biology techniques and methods. The resulting expressed antibody therapeutic is processed and purified according to state of the art biotechnology s, whether ly available or confidentially maintained by the owners of the composition of matter for the dy therapeutic. Methods to obtain the desired purified antibody product include ion exchange chromatography.

Example 5 Isolation of adult (somatic) antibody ing cells for reprogramming and transfection to produce biologic antibody therapeutics with redifferentiation to somatic antibody producing cells.

Antibody producing B cells are isolated from eral blood, bone marrow and other readily accessible hematopoietic cell s for the purpose of generating t specific manufacturing cell lines to produce eutic antibody biologics. Limiting dilution or cell sorting methods may be employed to select cells producing the highest levels of immunoglobulin (lg). After brief expansion, the clonal high Ig producing cell is reprogrammed to a pluripotent or progenitor state using standard reprogramming techniques.

The resulting patient specific stem cells are uced with the d antibody gene constructs using standard techniques and methods. Following gene modification, the pluripotent patient specific cell line is differentiated to a mature antibody producing B cell by culture in the ce of CD40L, BAFF, toll-like receptor activation (TLR) [Hayashi BA, el al. “TLR4 promotes B cell maturation: ndence and cooperation with B lymphocyte- activating factor”., J Immunol., [84:4662-4672 (2010), or other B cell maturation as is known in the art such as B-cell receptor (BCR) activation and Notch-receptor ligand family activation [Palanichamy A. at al. “Novel human transitional B cell populations revealed by B cell depletion therapy”. 10, May 2009,] Immunol, Vol. 182, pp. 5982-5993 ; Thomas MD. at 0]., “Regulation of peripheral B cell maturation”., Cell Ilium/1101., 239:92-102 (2006’).

Titers and affinity of the therapeutic antibody can be improved by the use of s such as morphogenic techniques as described Li J., at 01., “Human dies for immunotherapy development generated via a human B ceil hybridoma technology”., I’roc Natl Acad Sci , [033557-3562 (2006). The resulting expressed antibody therapeutic is processed and purified according to state of the art biotechnoiogy methods, whether publicly available or confidentially maintained by the owners of the composition of matter for the antibody therapeutic.

Alternatively, the pluripotent patient specific cell line is differentiated to a mature dy producing B cell by culture in the presence of CD40L, BAFF, toll-like or tion (TLR) (See Hayashi, er 0/. id)., or other B cell maturation factor as is known in the art such as B-cell receptor (BCR) activation and Notch—receptor ligand family activation. (See Palanichamy A. er al. 1761., and Thomas, MD, at 0/. id.) Titers and affinity of the therapeutic antibody can be improved by the use of methods such as morphogenic techniques as described (See Li, et a]. id.) The resulting patient ic antibody producing cells are transduced with the desired antibody gene constructs using standard techniques and methods. The resulting expressed antibody therapeutic is processed and purified according to state of the art hnology methods, whether publicly ble or ntially maintained by the owners of the composition of matter for the antibody therapeutic.

Example 6 Generation of patient specific cell lines for production of high ty ADCC antibodies.

N-acetylglucosamine (GlcNac) post-translational modification of immunoglobulins is important for antibodyvdependent cell-mediated toxicity (ADCC), and cosylated GlcNac residues have the highest affinity for Fc gamma receptors Mori K., e/ a/., “Non-fucosylated therapeutic antibodies: the next generation of therapeutic dies”., Cit/oraclmologvu 55: [09-1 14 (2007). ore, when an dy therapeutic with high levels of ADCC is desired, a patient specific cell line capable of transferring GlcNac at the appropriate levels and leaving the GlcNac non-fucosylated is desirable. Carcinoma ceils are known to express higher levels of OMB and therefore, since cancer stem celis and pluripotent cells have similar genetic signatures, pluripotent cells may also be suspected to express high levels of GMD, the enzyme responsible for post-translational GlcNac attachment. Among normal s, colon and pancreas express the highest levels of GMD. Lack of FUT8, which is responsible for the enzyme that fucosylates antibodies would be desirable in a patient specific stem cell line for therapeutic dy production dependent on ADCC. activity for efficacy, such as Rituximab or Herceptin. For instance, monoclonal antibodies produced in rat hybridoma YBZ/O cells have 50 fold higher ADCC activity than the same monoclonal antibodies produced using CHO cells. Adipose-derived stem cells and germ cell lines, as well as B cell lymphomas, express higher than average levels of FUTS, while hematopoietic stem cells (H SC), immature B cells, normal skeletal muscle express lower than e FUTS. poietic stem cells are ed according to rd methods from bone marrow aspirates, or whole blood apheresis with or without prior treatment with stem cell mobilizing agents. The isolated HSC are uently reprogrammed to pluripotency as previously described. The resulting patient specific stem cells are transduced with the desired antibody gene ucts using standard techniques and methods. Following gene transfection, the pluripotent patient specific cell line is differentiated to a mature antibody producing B cell by culture in the presence of CD40L, BAFF, toll-like receptor tion (TLR) (See Hayashi E.A., el 01., id), or other B cell maturation factor as is known in the art such as B-cell receptor (BC'R) activation and Notch-receptor ligand family activation (See Palanichamy A. at 0]. id, and Thomas, MD. et 01. id.) Titers and y of the therapeutic antibody can be improved by the use of methods such as morphogenic techniques as described (See Li, et a]. id.) The resulting expressed antibody eutic is processed and ed according to state of the an biotechnology methods, whether publicly available or confidentially maintained by the owners of the composition of matter for the antibody therapeutic.

Alternatively, antibody producing B ceils are isolated from peripheral blood, bone marrow and other readily accessible hematopoietic cell sources for the purpose of generating patient specific manufacturing cell lines to produce therapeutic antibody biologics.

Limiting dilution or cell sorting methods may be employed to select cells producing the highest levels of immunoglobulin (lg). After brief expansion, the clonal high lg ing cell is reprogrammed to a pluripotent or progenitor state using standard ramming techniques. The resulting patient specific stem cells are transduced with the desired antibody gene ucts using standard techniques and methods. Following gene modification, the pluripotent patient specific cell line is entiated towards an HSC, immature B cell or skeletal muscle cell for production of therapeutic antibodies that are low or lacking fucose.

While the prefeired embodiment of the invention has been illustrated and described, as noted ab0ve, 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 sure of the preferred embodiment. Instead, the invention should be determined entirely by reference to the claims that follow.

BRIEF DESCRIPTION OF THE DRAWINGS Preferred and alternative examples of the t invention are described in . detail below with reference to the ing drawings: Figure one depicts the layers resulting from gradient centrifugation of whole blood.

Claims (19)

1. A method for producing a therapeutic recombinant biologic polypeptide or protein for treating a disease comprising transfecting a synthetically-produced Pluripotent Stem Cell (spPSC) with a nucleic acid that encodes for said therapeutic recombinant biologic ptide or protein, under conditions wherein the polypeptide or protein is expressed by said spPSC, wherein the spPSC is produced from a cell of an animal that is not within a human body.

2. The method of claim 1 wherein the therapeutic biologic polypeptide or protein is selected from the group ting of erythropoietin, factor VIII, factor IX, thrombin, an antibody or antibody fragment, alpha interferon, interferon alpha 2A and 2B, beta interferon, growth hormone, antihemophilic factor, G-CSF, GM-CSF, a soluble receptor, TGF-ß, bone morphogenic proteins (BMP), TGF, interleukin 2, ß-glucocerebrosidase or an analogue thereof, alpha1-proteinase inhibitor, fibrin, fibrinogen, von Willebrand factor, imiglucerase, dase beta, laronidase, glucosidase alpha, thyrotropin alpha, and thymosin alpha.

3. The method of claim 1 or claim 2 wherein the antibody or antibody fragment binds to a target, n said target is selected from the group ting of a tumor is factor (TNF) molecule, a tumor necrosis factor receptor (TNFR), a growth factor receptor, a vascular endothelial growth factor (VEGF) molecule, interleukin 1, interleukin 4, interleukin 6, interleukin 11, interleukin 12, gamma interferon, receptor tor of nuclear factor kappa-B ligand (RANKL) and Blys.

4. The method of claim 2 n said soluble or binds to a target selected from the group consisting of TNF, TNF and Blys.

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

6. The method of claim 5 further comprising inducing differentiation of the ected, immortalized .

7. The method of any one of claim 1 further comprising inducing differentiation of the transfected spPSCs. AH26(10206567_1):CCG [Link] http://en.wikipedia.org/wiki/Monoclonal_antibodies

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

9. The method of any one of claims 1 to 8 n the spPSC is selected from the group consisting of ected non-Pluripotent Stem Cells, pluripotent stem cells produced by Somatic cell nuclear transfer (SCNT pluripotent stem cells), pluripotent stem cells produced by Altered nuclear transfer, oocyte assisted reprogramming (ANT-OAR pluripotent stem cells) and pluripotent stem cells produced by parthenogenesis (PGA pluripotent).

10. The method of any one of claims 1 to 9 wherein the therapeutic recombinant biologic polypeptide or protein is selected from the group consisting of: growth hormones, cytotoxic biologics, and monoclonal antibodies.

11. Use of a therapeutic recombinant biologic polypeptide or protein ed from a tically-produced Pluripotent Stem Cell (spPSC) d from the species of an animal suffering from a disease, in the preparation of a medicament for treating said disease in the animal, n the spPSC has been transfected with a nucleic acid that encodes for the therapeutic recombinant biologic polypeptide or protein.

12. The use of claim 11 wherein the eutic biologic polypeptide or protein is selected from the group consisting of erythropoietin, factor VIII, factor IX, thrombin, an antibody or antibody fragment, alpha interferon, interferon alpha 2A and 2B, beta interferon, growth hormone, antihemophilic factor, G-CSF, GM-CSF, a soluble receptor, TGF-ß, bone genic proteins (BMP), TGF, interleukin 2, ß-glucocerebrosidase or an analogue thereof, alpha1-proteinase tor, fibrin, fibrinogen, von Willebrand factor, imiglucerase, dase beta, laronidase, glucosidase alpha, thyrotropin alpha, and thymosin alpha.

13. The use of claim 11 or claim 12 further comprising immortalizing the transfected spPSC.

14. The use of claim 13 further comprising inducing differentiation of the transfected, immortalized spPSCs. 10099616 [Link] http://en.wikipedia.org/wiki/Monoclonal_antibodies

15. The use of claim 11 or claim 12 further comprising inducing differentiation of the transfected spPSC.

16. The use of claim 15 further comprising immortalizing the transfected, differentiated cells.

17. The use of any one of claims 11 to 16 wherein the spPSC is ed from the group consisting of: transfected non-Pluripotent Stem Cells, pluripotent stem cells produced by c cell nuclear transfer (SCNT pluripotent stem cells), otent stem cells produced by Altered nuclear transfer, oocyte assisted reprogramming (ANT-OAR otent stem cells) and pluripotent stem cells produced by parthenogenesis (PGA pluripotent).

18. The use of any one of claims 11 to 17 wherein the eutic recombinant ic polypeptide or protein is selected from the group consisting of: growth hormones, cytotoxic biologics, and monoclonal antibodies.

19. A therapeutic recombinant biologic polypeptide or protein for treating a disease produced by the method of any one of claims 1 to 10.