WO2016183593A2 - Prenatal therapy - Google Patents

Abstract

Compositions and methods are provided to treat or prevent a genetic disease or disorder, e.g., Hemophilia A, in a subject by administering to the subject an effective amount of a polynucleotide encoding a nucleic acid encoding a functional Factor VIII polypeptide in a pre-term placenta-derived stem cell. The subject to be treated can be a fetus and the cell is a pre-term chorionic villus tissue derived stem cell (CSC) that has been isolated from the placenta before the baby is born.

Description

PRENATAL THERAPY

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority under 35 U.S.C. § 1 19(e) to U.S. Provisional Application No. 62/161 ,793, filed May 14, 2015, the content of which is hereby incorporated by reference in its entirety.

BACKGROUND

[0002] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation and in some aspects, the complete bibliographic of the citation is found in the section immediately preceding the claims. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.

[0003] Hemophilia A (HA) is an X-linked recessive bleeding disorder caused by a mutation in the gene encoding Factor VIII (FVIII). Patients with severe HA suffer from debilitating hemarthroses, life-threatening internal bleeding, and potentially fatal intracranial hemorrhage. The current standard of care for HA is FVIII protein substitution therapy (PST), which offers reliable prophylactic and therapeutic relief from bleeding episodes. However, PST is not a cure, is prohibitively expensive, and is unavailable to -75% of HA patients. In addition, 30- 40% of HA patients who undergo PST develop inhibitory antibodies to FVIII, rendering PST ineffective, increasing morbidity/mortality and drastically increasing the cost of treatment. Novel therapies to achieve sustained FVIII expression in HA patients are needed to overcome these limitations. This disclosure satisfies this need and provides related advantages as well.

SUMMARY

[0004] This disclosure provides methods to treat or prevent genetic diseases or disorders, for example Hemophilia A, a lysosomal storage disorder (LSD), Duchenne's Muscular Dystrophy, osteogenesis imperfecta (OI), thalassemia, sickle cell anemia, systic fibrosis, spinal muscular atrophy, or severe intrauterine growth restriction, in a subject in need thereof, comprising, or alternatively consisting essentially of, or yet further consisting of,

administering to the subject an effective amount of a nucleic acid encoding a therapeutic, functional polypeptide, e.g., a Factor VIII polypeptide for the treatment of Hemophilia A, suflimadase for MPS Ilia, (a LSD) or dystrophin for the treatment of DMD. In one aspect, the disease to be treated is Hemophilia A and the protein is Factor VIII polypeptides. Non- limiting examples of sFactor VIII polypeptides include wildtype Factor VIII, a fragment thereof, a B-domain deleted Factor VIII polypeptide (FVIII) or an equivalent thereof, a chimeric polypeptide encoding functional Willebrand factor polypeptide, or a fusion polypeptide, or an equivalent thereof. A non-limiting example of a polynucleotide encoding Factor VIII is provided in SEQ ED NO.: 2 from about 5253 to about 9626, or an equivalent thereof. The nucleic acid is administered in a cell isolated from placenta, such as a pre-term placenta-derived stem cell (also referred to chorionic villus sampling tissue derived stem cells ("CSCs") isolated from the mother carrying the fetus. Non-limiting examples of cells to transfer the nucleic acid include mesenchymal stem cells (C-MSCs), hemaptoieitc stem cells (C-HSCs), epithelial progenitor cells, or endothelial progenitor cells that also can be isolated from this tissue. Examples also include other cells, such as endothelial cells, that can be derived or differentiated from these pre-term placenta-derived stem cells. As used herein, the term "pre-term placenta-derived stem cell" intends a cell isolated from placental tissue prior to delivery of the fetus by surgery or birth. In another aspect, the isolated cell is a pre-term chorionic villus tissue derived stem cell (CSC) comprising the nucleic acid encoding the functional Factor VIII polypeptide. In an alternate embodiment, the isolated cell further comprises a nucleic acid encoding functional von Willebrand factor.

[0005] The method of this disclosure is not limited by which the nucleic acid encoding the therapeutic protein is inserted into a pre-term placenta-derived cell. Non-limited examples include vector-mediated insertion, CRISPR (CRISPR-Cas9) or TALENs.

[0006] In one aspect, the nucleic acid encoding functional therapeutic polypeptide and/or the nucleic acid encoding a functional, therapeutic protein is contained within a vector that is used to transduce the cell with the nucleic acid(s). The vectors can further comprise a promoter operatively linked to the nucleic acids. Non-linking examples of promoters to drive expression of the nucleic acids include an MDNU3 promoter or a PGK promoter. In another aspect, the vector further comprises one or more enhancer elements. In a further aspect, the polynucleotide encodes functional von Willebrand factor which can be in a separate vector and transduced into the same cell or another isolated cell for coadministration to the subject in need of this therapy.

[0007] In one aspect, the compositions and methods described herein are useful in the treatment of a fetus, such as a human fetus. In one aspect, the cells to be transduced with the nucleic acids and in one aspect, the vectors described herein are first isolated from the early gestational placenta, in one aspect, the mother carrying the fetus to be treated. The cells are then transduced with the nucleic acid and/or a vector comprising the nucleic acid the therapeutic, functional protein. In one aspect, the isolated, transduced cells are expanded to a population of cells containing the nucleic acids prior administration to the subject, e.g., the fetus. The patient or subject is treated by expression of the functional protein in the subject in, or by a cell in subject in need of such treatment. Methods to determine if functional protein is being expressed are known in the art, e.g., by the use of polymerase chain reaction, histology and the use antibodies that bind functional protein. In addition, and after birth of the fetus, the child can be monitored for clinical symptoms of the genetic disorder. A reduction or complete elimination of clinical symptons are alternative indications of effective therapy. The therapeutic methods as described herein can be combined with a pre-term diagnostic to determine if the fetus is likely to develop the genetic disease or disorder, e.g., Hemophilia A, prior to the administration of the therapy. Such methods are known in the art.

[0008] This disclosure also provides the compositions to prepare the cells for the therapy. In one aspect, a vector is provided that is used to transduce the isolated cells used for the therapy, such as an isolated CSC. The vector comprises: (a) a backbone comprising essential sequences for integration into a target cell genome; (b) a nucleic acid encoding a therapeutic, functional polypeptide; and (c) a first expression control element that regulates expression of the nucleic acid encoding the functional polypeptide. In another aspect, the vector further comprises (d) a nucleic acid encoding a marker polypeptide and (e) a second control element that regulates expresson of the nucleic acid encoding the marker polypeptide. In a further aspect, the vector futher comprises (e) a nucleic acid encoding functional polypeptide and (f) a second control element that regulates expression of the functional polypeptide. In a yet further aspect, the vector further comprises (g) one or more enhancer elements. In a yet further aspect, the vector further comprises (h) a P2A an internal protease coding site, wherein P2A is the nucleotide sequence encoding a peptide that promotes a ribosomal skip resulting in the stoichiometric expression of two unfused reporter proteins from the same mRNA transcript. In further aspects, the disclosure provides vector comprises: (a) a backbone comprising essential sequences for integration into a target cell genome; (b) a nucleic acid encoding a functional polypeptide; and (c) an expression control element that regulates expression of the nucleic acid encoding the functional polypeptide. The vector can further comprise a nucleic acid encoding a marker polypeptide under the control of a promoter element. The vector can further comprise an enhancer element. In one aspect the vectors are viral vectors, such as lentiviral vectors. [0009] In one embodiment, the one or more expression control elements are Polymerase II promoters, non-limiting examples of such are MNDU3 and phosphoglycerate kinase (PGK) promoter. Non-limiting examples of detectable markers are Green Flourescent Protein (GFP) or luciferase (LUC). A non-limiting example of an enhancer element is WPRE. A non- limiting example of a vector comprising these elements is provided in SEQ ID NOs: 1 or 2 or a polynucleotide having at least 80 % identity thereto.

[0010] This disclosure also provides a viral packaging system comprising: (a) the vector(s) as described above wherein the backbone is derived from a virus; (b) a packaging plasmid; and (c) an envelope plasmid. In one aspect, the envelope plasmid is a plasmid comprising a S. aureus ZZ domain sequence, or a sequence encoding a pseudotyped envelope protein or a VSVG envelope, or a pMD.G VSV sequence or other envelope plasmids known in the art and available from addgene (addgene.org/12259, last accessed on May 12, 2015). Also provided herein is a packaging cell line such as HEK-293 cell for viral production.

[0011] Further provided herein is a method for producing a pseudotyped viral particle that expresses a functional polypeptide by transducing one or more packaging cell lines as described above with the system as described above under conditions suitable to package the viral vector, as well as the pseudotyped viral particle produced by the methods.

[0012] As noted above, the pseudotyped viral particle is useful to transduce an isolated cell such as a CSC cell which in turn is useful for the treatment or prevention of the genetic disease in a subject in need thereof. Thus, this disclosure also provides the pseudotyped viral particle conjugated or attached to the isolated cell to be infected by the virus. Accordingly, also provided is the isolated cell comprising one or more of: the nucleic acids and/or the vectors and/or the pseudotyped viral particles as described herein.

[0013] Also as noted above, the compositions are useful in a therapeutic method to treat a fetus determined to be at risk of developing Hemophilia A upon birth and the nucleic acid encoding functional polyeptide is contained within the cell, such as a stem cell isolated from pre-term placental tissue. Non-limiting example of such cells include mesenchymal stem cells (C-MSCs), hemaptoieitc stem cells (C-HSCs), epithelial progenitor cells, and endothelial progenitor cells.Applicants have determined that C-MSCs that are useful in the method express one or more of the markers CD105, CD90, CD73, CD44, and CD29 and did not express one or both of CD184 and/or HLA-DR. In addition or alternatively, the C-MSCs do not express the markers CD45, CD34 and/or CD31 and in one aspect, are isolated from early gestation placenta.

[0014] Also provided is a population of C-MSCs as described herein. Such population can be a substantially homogenous population of cells such as a clonal population. Alternatively, the stem cells can be expanded and differentiated into a population of one or more mesodermal lineages, e.g. osteogenic cell, an adipogenic cell or a chondrogenic cell.

[0015] Any one or more of the cells, vectors, packaging systems, and/or pseudotyped viral particles can be combined with a carrier, such as a pharmaceutically acceptable carrier.

[0016] Yet further provided is a method to express a functional polypeptide by culturing the cell as described above under conditions that favor expression of the functional polypeptide.

[0017] Yet further provided is a method for forming a matrix, comprising, or alternatively consisting essentially of, or yet further consisting of, combining a population of cells as described above with a pharmaceutically acceptable carrier such as a biocompatible matrix or scaffold, optionally suitable for implantation in vivo e.g., in utero.

[0018] Kits are also provided, having the compositions described above, alone or in combination, and optionally, reagents and instructions for use of one or more of:

diagnostically, as a research tool or therapeutically.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIGS. 1A-1C show CVS as a feasible means of deriving mesenchymal stem cells (C-MSCs) for therapeutic purposes. (FIG. 1A) Example of average CVS-sized tissue size and mass. (FIG. IB) Mean and standard deviation of chorionic villus tissue masses, cell counts at each passage, and days in vitro (DIV) between passages. (FIG. 1C) Growth curves representing the first 3 passages of cells obtained from CVS-sized samples from six placentas of varying gestational age ( 12- 1 8 weeks).

[0020] FIGS. 2A-2B show phenotypic characterization of C-MSCs. (FIG. 2A) Flow cytometric analysis showing MSC markers. (FIG. 2B) C-MSC trilineage differentiation potential (Adipogenic, Chondrogenic and Osteogenic). Scale Bar = 100 μπι.

[0021] FIGS. 3A-3B show FVIII expression in transduced C-MSCs. (FIG. 3A)

Fluorescence imaging of C-MSCs transduced with an expression vector for FVIII tagged with GFP. (FIG. 3B) ~ 90% FVIII-GFP expression in flow cytometric analysis of transduced C- MSCs. Scale Β3Γ=100μηι. [0022] FIG. 4 shows immunocytochemistry detection of human FVIII (hFVIII) protein in C-MSCs after lentiviral-mediated transduction. C-MSCs transduced with an expression vector to FVIII (red), GFP (green) and DAPI (blue) for the nuclei. Scale Β3τ=100μηι.

[0023] FIGS. 5A-5B show bioluminescence image (BLI) analysis. BLI of gravid dam (FIG. 5A) showing focal density of C-MSCs transplanted in the fetuses and in the 5-day-old pup (FIG. 5B) born from the dam 12 days after in utero transplantation of C-MSCs.

[0024] FIG. 6 shows optimizing the transduction of C-MSCs using the new vectors. C- MSCs (cell line #450 as a representative) were transduced with either X-NEO or FVIII-NEO at different MOI and incubated for 72h. FVIII activity in the media was assessed using chromogenic assay.

[0025] FIG. 7 shows four cell lines of C-MSCs were transduced with either X-NEO or FVIII-NEO at a MOI of 10 and incubated for 72h. Expression of FVIII was assessed by RT- PCR.

[0026] FIG. 8 shows four cell lines of C-MSCs were transduced with either X-NEO or FVIII-NEO at a MOI of 10 and incubated for 72h. Expression of FVIII was assessed by Western blotting.

[0027] FIG. 9 shows four cell lines of C-MSCs were transduced with either X-NEO or FVIII-NEO at a MOI of 10 and incubated for 72h. FVIII activity in the media was assessed using chromogenic assay.

BRIEF DESCRIPTION OF SELECTED SEQUENCE LISTINGS

[0028] SEQ ID NO: 1 is the sequence of the vector : pCCLc-MNDU3 -Factor VIII ("F8").

[0029] SEQ ID NO: 2 is the sequence of the vector : pCCLc-MNDU3 -Factor VIII ("F8")- WPRE.

[0030] SEQ ID NO: 3 is the sequence of an exemplary packaging plasmid - pCMVdR8.91.

[0031] SEQ ID NO: 4 is the sequence of an exemplary envelope plasmid pMDG-VSVG.

[0032] SEQ ID NO: 5 is the sequence of the vector: pCCLc-MNDU3c-Factor VIII ("F8")- PGK-LUC-P2A-EGFP. MODES FOR CARRYING OUT THE INVENTION

Definitions

[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0034] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook and Russell eds. (2001 ) Molecular Cloning: A Laboratory Manual, 3^rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1 : A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Techique, 5^th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid

Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press ( 1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987)

Immunochemical Methods in Cell and Molecular Biology (Academic Press, London);

Herzenberg et al. eds (1996) Weir's Handbook of Experimental Immunology; Manipulating the Mouse Embryo: A Laboratory Manual, 3^rd edition (Cold Spring Harbor Laboratory Press (2002)); Sohail (ed.) (2004) Gene Silencing by RNA Interference: Technology and

Application (CRC Press).

[0035] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, including ranges, are approximations which are varied ( + ) or ( - ) by increments of 0.1 or 1.0, where appropriate. It is to be understood, although not always explicitly stated that all numerical designations are preceded by the term "about." It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0036] As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0037] As used herein, the term "comprising" or "comprises" is intended to mean that the compositions and methods include the recited elements, but not excluding others.

"Consisting essentially of when used to define compositions and methods, shall mean excluding other elements of any essential significance to the combination for the stated purpose. Thus, a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and

pharmaceutically acceptable carriers, such as phosphate buffered saline, preservatives and the like. "Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention or process steps to produce a composition or achieve an intended result. Embodiments defined by each of these transition terms are within the scope of this invention.

[0038] The term "isolated" as used herein with respect to nucleic acids, such as DNA or R A, refers to molecules separated from other DNAs or R As, respectively that are present in the natural source of the macromolecule. The term "isolated nucleic acid" is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state. The term "isolated" is also used herein to refer to polypeptides, proteins and/or host cells that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. In other embodiments, the term "isolated" means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature. For example, an isolated cell is a cell that is separated form tissue or cells of dissimilar phenotype or genotype. As is apparent to those of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, does not require "isolation" to distinguish it from its naturally occurring counterpart. [0039] The terms "polynucleotide", "nucleic acid" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (for example, a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A polynucleotide can comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. A polynucleotide can be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of this invention that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0040] A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term "polynucleotide sequence" is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be input into databases in a computer having a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.

[0041] As used herein, the term "genetic disease or disorder" intends a disease caused in whole or in part by a change in the DNA sequence away from the normal sequence. As defined by the National Human Genome Research Institute (genome.gov), genetic disorders can be caused by a mutation in one gene (monogenic disorder), by mutations in multiple genes (multifactorial inheritance disorder), by a combination of gene mutations and environmental factors, or by damage to chromosomes (changes in the number or structure of entire chromosomes, the structures that carry genes). As used herein, the genetic diseases include inherited or acquired from during a patient's life. Non-limited examples of genetic disorders include sickle cell disease, DMD, LSDs, cystic fibrosis, Tay-Sachs disease, Hemophilia A, osteogenesis imperfecta (01), thalassemia, spinal muscular atrophy, severe intrauterine growth restriction, or MPS.

[0042] A lysosomal storage disorders or "LSDs" are a group of approximately 50 rare inherited metabolic disorders that result from defects in lysosomal function. Lysosomal storage disorders are caused by lysosomal dysfunction usually as a consequence of deficiency of a single enzyme required for the metabolism of lipids, glycoproteins (sugar containing proteins) or so-called mucopolysaccharides. Individually, LSDs occur with incidences of less than 1 : 100,000; however, as a group the incidence is about 1 :5,000 - 1 : 10,000. Most of these disorders are autosomal recessively inherited such as Niemann-Pick disease, type C, however a few are X-linked recessively inherited, such as Fabry disease and Hunter syndrome (MPS II).

[0043] Mucopolysaccharidosis type III (MPS ΠΙ), also known as Sanfilippo syndrome, is a progressive disorder that mainly affects the brain and spinal cord (central nervous system). Patients with MPS III generally do not display any features of the condition at birth, but they begin to show signs and symptoms of the disorder during early childhood. Affected children often initially have delayed speech and behavior problems. They may become restless, destructive, anxious, or aggressive. Sleep disturbances are also very common in children with MPS III. This condition causes progressive intellectual disability and the loss of previously acquired skills (developmental regression). In later stages of the disorder, people with MPS III may develop seizures and movement disorders. MPS III is divided into types IIIA, IIIB, IIIC, and HID, which are distinguished by their genetic cause. The different types of MPS III have similar signs and symptoms, although the features of MPS ΠΙΑ typically appear earlier in life and progress more rapidly. People with MPS III usually live into adolescence or early adulthood. MPS III includes 4 types, each due to the deficiency of a different enzyme:

heparan N-sulfatase (type A); alpha-N-acetylglucosaminidase (type B;); acetyl CoA:alpha- glucosaminide acetyltransferase (type C;); and N-acetylglucosamine 6-sulfatase (type D).

[0044] Duchenne's muscular dystrophy (DMD) is an X-linked recessive form of muscular dystrophy, affecting around 1 in 3,600 boys, which results in muscle degeneration and premature death. The disorder is caused by a mutation in the gene dystrophin, located on the human X chromosome, which codes for the protein dystrophin. Dystrophin is an important component within muscle tissue that provides structural stability to the dystroglycan complex (DGC) of the cell membrane. While both sexes can carry the mutation, females are rarely affected. The polynucleotide (mRNA) and protein sequences for dystrophin are available at NM_000109.3 and NP_000100.2, respectively.A clinical test of DMD is the six minute walk test.

[0045] Factor VIII (FVIII) is an essential blood-clotting protein also known as antihemophilic factor (AHF). In humans, factor VIII is encoded by the F8 gene Defects in this gene results in hemophilia A, a recessive X-linked coagulation disorder. Factor VIII circulates in the bloodstream in an inactive form, bound to another molecule called von Willebrand factor, until an injury that damages blood vessels occurs. In response to injury, coagulation factor VIII is activated and separates from von Willebrand factor. The active protein is sometimes called coagulation factor Villa.

[0046] As used herein, the term "functional Factor VIII (FVIII) polypeptide intends a polypeptide from any species (e.g., a mammalian, a human, an ovine, a bovine, an equine, a canine, a feline or a murine). Non-limiting examples of nucleic acids encoding functional Factor VIII polypeptides include without limitation: a nucleic acid encoding wild-type polypeptide and equivalents thereof; a nucleic acid encoding B-domain deleted Factor VIII polypeptide or an equivalent thereof, or a nucleic acid encoding a fragment of Factor VIII polypeptide or an equivalent thereof, that when administered to a subject, corrects for Hemophilia A, including fusion proteins comprising such fragments or equivalents thereof. An additional non-limiting example of such is the B-domain deleted FVIII, and

polynucleotides encoding this polypeptide or an equivalent thereof. Additional examples include polynucleotides found under Accession No. NP_000123 (human), mRNA encoding the protein is NM_000132 (human), and the sequences provided in the Sequence Listing attached hereto, and incporated herein by reference.. The mouse protein is found under NP_001 154845 and mRNA encoding it is found under NM_001 161373.

[0047] As used herein, the term "functional von Willebrand factor polypeptide intends a polypeptide from any species (e.g., a mammalian, a human, an ovine, a bovine, an equine, a canine, a feline or a murine). Non-limiting examples of nucleic acids encoding functional von Willebrand factor include without limitation a nucleic acid encoding the wild-type polypeptide or an equivalent thereof, or a nucleic acid encoding a fragment of von Willbrand factor or an equivalent thereof, fusion proteins containing a fragment or an equivalent thereof and modified polypeptides that when administered have the same or similar biological activity to wild-type von Willebrand factor. [0048] Polynucleotides encoding von Willebrand factor, fragments, modified versions and equivalents thereof and Factor VIII factor, fragments, modified versions (such as fusion polypeptides, e.g., fused to XTEN or albumin), and equivalents thereof, are known in the art and disclosed in U.S. Patent Application Publication Nos. 2014/0357564; 2014/0072561 ; 2014/0056861 ; 2013/0017997 (Factor VIII polypeptide linked to (extended recombinant polypeptide (XTEN)); 2013/0296244, 2013/0108629 (Factor VIII- Fc chimeric and hybrid polypeptides); and 2013/0267468. The polynucleotides and polpeptides are from any species (e.g., a mammalian, a human, a ovine, a bovine, an equine, a canine, a feline or a murine).

[0049] A "marrow stromal cell" also referred to as "mesenchymal stem cells," or MSC, is a multipotent stem cell that can differentiate into a variety of cell types. Cell types that MSCs have been shown to differentiate into in vitro or in vivo include osteoblasts, chondrocytes, myocytes, and adipocytes. Mesenchyme is embryonic connective tissue that is derived from the mesoderm and that differentiates into hematopoietic and connective tissue, whereas MSCs do not differentiate into hematopoietic cells. Stromal cells are connective tissue cells that form the supportive structure in which the functional cells of the tissue reside. Methods to isolate such cells, propogate and differentiate such cells are known in the technical and patent literature, e.g., U.S. Patent Application Publication Nos. 2007/0224171 , 2007/0054399 and 2009/0010895, which are incorporated by reference in their entirety.

[0050] "CSCs" is an acronym for chorionic villus tissue derived stem cells. "pMSCs" or "PMSCs" or "mpSCs" or "C-MSCs"are acronyms for mesenchymal stem cells isolated or purified from placental tissue prior to delivery of the fetus by surgery or birth. Within this disclosure, the cells also are referred to as pre-term placenta-derived stem cell (mpSCs) or when isolated by chorionic villus sampling, they are identified as C-MSCs. In one aspect, the C-MSCs express angiogenic and immunomodulatory cytokines (e.g., Angiogenin,

Angiopoietin- 1 , HGF, VEGF, IL-8, MCP- 1 , uPA).

[0051] The term "propagate" means to grow or alter the phenotype of a cell or population of cells. The term "growing" refers to the proliferation of cells in the presence of supporting media, nutrients, growth factors, support cells, or any chemical or biological compound necessary for obtaining the desired number of cells or cell type. In one embodiment, the growing of cells results in the regeneration of tissue. In yet another embodiment, the tissue is comprised of neuronal progenitor cells or neuronal cells. [0052] The term "culturing" refers to the in vitro propagation of cells or organisms on or in media of various kinds. It is understood that the descendants of a cell grown in culture may not be completely identical (i.e., morphologically, genetically, or phenotypically) to the parent cell. By "expanded" is meant any proliferation or division of cells. A "cultured" cell is a cell that has been separated from its native environment and propagated under specific, predefined conditions.

[0053] "Differentiation" describes the process whereby an unspecialized cell acquires the features of a specialized cell such as a heart, liver, or muscle cell. "Directed differentiation" refers to the manipulation of stem cell culture conditions to induce differentiation into a particular cell type. "Dedifferentiated" defines a cell that reverts to a less committed position within the lineage of a cell. As used herein, the term "differentiates or differentiated" defines a cell that takes on a more committed ("differentiated") position within the lineage of a cell. As used herein, "a cell that differentiates into a mesodermal (or ectodermal or endodermal) lineage" defines a cell that becomes committed to a specific mesodermal, ectodermal or endodermal lineage, respectively. Examples of cells that differentiate into a mesodermal lineage or give rise to specific mesodermal cells include, but are not limited to, cells that are adipogenic, leiomyogenic, chondrogenic, cardiogenic, dermatogenic, hematopoetic, hemangiogenic, myogenic, nephrogenic, urogenitogenic, osteogenic, pericardiogenic, or stromal.

[0054] Examples of cells that differentiate into ectodermal lineage include, but are not limited to epidermal cells, neurogenic cells, and neurogliagenic cells.

[0055] Chorionic Villus Sampling (CVS) is a technique to diagnose complications during a pregnancy. A small section of placental tissue is collected without disturbing the pregnancy, and these cells are examined for disease. The tissue sampled is the chorionic villus, and CVS is the technique used to obtain chorionic villus samples.

[0056] An "enhancer" is a regulatory element that increases the expression of a target sequence. A "promoter/enhancer" is a polynucleotide that contains sequences capable of providing both promoter and enhancer functions. For example, the long terminal repeats of retroviruses contain both promoter and enhancer functions. The enhancer/promoter may be "endogenous" or "exogenous" or "heterologous." An "endogenous" enhancer/promoter is one which is naturally linked with a given gene in the genome. An "exogenous" or "heterologous" enhancer/promoter is one which is placed in juxtaposition to a gene by means of genetic manipulation (i.e., molecular biological techniques) such that transcription of that gene is directed by the linked enhancer/promoter. In one aspect, the enhancer is a Woodchuck post- regulatory element ("WPRE") (see, e.g., Zufferey, R. et al. (1999) J. Virol. 73(4):2886-2992) and equivalents thereof having enhancer function. A non-limiting example of a WPRE enhancer is found in the attached Sequence Listing, i.e., nucleotides 9757 to 10353 of Seq. ID NO: 2, or an equivalent thereof.

[0057] As is known to those of skill in the art, there are 6 classes of viruses. The DNA viruses constitute classes I and II. The RNA viruses and retroviruses make up the remaining classes. Class III viruses have a double-stranded RNA genome. Class IV viruses have a positive single-stranded RNA genome, the genome itself acting as mRNA Class V viruses have a negative single-stranded RNA genome used as a template for mRNA synthesis. Class VI viruses have a positive single- stranded RNA genome but with a DNA intermediate not only in replication but also in mRNA synthesis. Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.

[0058] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, or alternatively less than 25% identity, with one of the sequences of the present invention.

[0059] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + SwissProtein + SPupdate + PIR. Details of these programs can be found at the following Internet address: http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST.

[0060] An equivalent nucleic acid, polynucleotide or oligonucleotide is one having at least 80 % sequence identity, or alternatively at least 85 % sequence identity, or alternatively at least 90 % sequence identity, or alternatively at least 92 % sequence identity, or alternatively at least 95 % sequence identity, or alternatively at least 97 % sequence identity, or alternatively at least 98 % sequence identity to the reference nucleic acid, polynucleotide, or oligonucleotide, or alternatively an equivalent nucleic acid hybridizes under conditions of high stringency to a reference polynucleotide or its complement.

[0061] An equivalent polypeptide or protein is one having at least 80 % sequence identity, or alternatively at least 85 % sequence identity, or alternatively at least 90 % sequence identity, or alternatively at least 92 % sequence identity, or alternatively at least 95 % sequence identity, or alternatively at least 97 % sequence identity, or alternatively at least 98 % sequence identity to the reference polypeptide or protein, or alternatively an equivalent polypeptide or protein is one encoded by nucleic acid that hybridizes under conditions of high stringency to a polynucleotide or its complement that encodes the reference polypeptide or protein.

[0062] The expression "amplification of polynucleotides" includes methods such as PCR, ligation amplification (or ligase chain reaction, LCR) and amplification methods. These methods are known and widely practiced in the art. See, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202 and Innis et al., 1990 (for PCR); and Wu et al. (1989) Genomics 4:560-569 (for LCR). In general, the PCR procedure describes a method of gene amplification which is comprised of (i) sequence-specific hybridization of primers to specific genes within a DNA sample (or library), (ii) subsequent amplification involving multiple rounds of annealing, elongation, and denaturation using a DNA polymerase, and (iii) screening the PCR products for a band of the correct size. The primers used are oligonucleotides of sufficient length and appropriate sequence to provide initiation of polymerization, i.e., each primer is specifically designed to be complementary to each strand of the genomic locus to be amplified.

[0063] Reagents and hardware for conducting PCR are commercially available. Primers useful to amplify sequences from a particular gene region are preferably complementary to, and hybridize specifically to sequences in the target region or its flanking regions. Nucleic acid sequences generated by amplification may be sequenced directly. Alternatively the amplified sequence(s) may be cloned prior to sequence analysis. A method for the direct cloning and sequence analysis of enzymatically amplified genomic segments is known in the art.

[0064] A "gene" refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated.

[0065] The term "express" refers to the production of a gene product.

[0066] As used herein, "expression" refers to the process by which polynucleotides are transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the rnRNA in a eukaryotic cell.

[0067] A "gene product" or alternatively a "gene expression product" refers to the amino acid (e.g., peptide or polypeptide) generated when a gene is transcribed and translated.

[0068] "Under transcriptional control" is a term well understood in the art and indicates that transcription of a polynucleotide sequence, usually a DNA sequence, depends on its being operatively linked to an element which contributes to the initiation of, or promotes, transcription. "Operatively linked" intends the polynucleotides are arranged in a manner that allows them to function in a cell. In one aspect, this invention provides promoters operatively linked to the downstream sequences, e.g., functional Factor VIII and markers.

[0069] The term "encode" as it is applied to polynucleotides refers to a polynucleotide which is said to "encode" a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and/or translated to produce the mRNA for the polypeptide and/or a fragment thereof. The antisense strand is the

complement of such a nucleic acid, and the encoding sequence can be deduced therefrom.

[0070] Clustered regularly interspaced short palindromic repeats (CRISPR) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to a bacterial virus or plasmid. CRISPRs are found in approximately 40% of sequenced bacteria genomes and 90% of sequenced archaea. As used herein, CRISPR intends the gene editing technique used for altering the germline of humans, animals, and other organisms and modifying the genes of food crops. By delivering the Cas9 protein and appropriate guide RNAs into a cell, the organism's genome can be cut at any desired location. Tools to use the CRISPR system for gene editing are commercially available, see., e.g., Clontech and genscript.The CRISP-Cas9 system has been used to transfer functional FactorVIII into iPSCs cells. Park et al. (2015) Cell Stem Cell, Vol. 1 7:213-220.

[0071] Transcription activator-like effector nucleases (TALENs) are restriction

enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands). Transcription activator-like effectors (TALEs) can be engineered to bind practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations. The restriction enzymes can be introduced into cells, for use gene editing or for genome editing in situ, a technique known as genome editing with engineered nucleases. Alongside zinc finger nucleases and CRISPR/Cas9, TALEN is a prominent tool in the field of genome editing. TALENs has been successfully used to correct a Factor VIII genetic defect in an iPSC cell line. Park et al. (2014) PNAS, Vol. 1 1 1 (25):9253-9258).

[0072] A "probe" when used in the context of polynucleotide manipulation refers to an oligonucleotide that is provided as a reagent to detect a target potentially present in a sample of interest by hybridizing with the target. Usually, a probe will comprise a detectable label or a means by which a label can be attached, either before or subsequent to the hybridization reaction. Alternatively, a "probe" can be a biological compound such as a polypeptide, antibody, or fragments thereof that is capable of binding to the target potentially present in a sample of interest.

[0073] "Detectable labels" or "markers" include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes.

Detectable labels can also be attached to a polynucleotide, polypeptide, antibody or composition described herein. In another aspect, the detectable marker is a protein or polypeptide expressed from a nucleic acid.

[0074] A "primer" is a short polynucleotide, generally with a free 3 ' -OH group that binds to a target or "template" potentially present in a sample of interest by hybridizing with the target, and thereafter promoting polymerization of a polynucleotide complementary to the target. A "polymerase chain reaction" ("PCR") is a reaction in which replicate copies are made of a target polynucleotide using a "pair of primers" or a "set of primers" consisting of an "upstream" and a "downstream" primer, and a catalyst of polymerization, such as a DNA polymerase, and typically a thermally-stable polymerase enzyme. Methods for PCR are well known in the art, and taught, for example in MacPherson et al. (1991 ) PCR 1 : A Practical Approach (IRL Press at Oxford University Press). All processes of producing replicate copies of a polynucleotide, such as PCR or gene cloning, are collectively referred to herein as "replication." A primer can also be used as a probe in hybridization reactions, such as Southern or Northern blot analyses. Sambrook and Russell (2001 ), infra.

[0075] "Hybridization" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi-stranded complex, a single self-hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of a PCR reaction, or the enzymatic cleavage of a polynucleotide by a ribozyme.

[0076] Hybridization reactions can be performed under conditions of different "stringency". In general, a low stringency hybridization reaction is carried out at about 40 °C in 10 x SSC or a solution of equivalent ionic strength/temperature. A moderate stringency hybridization is typically performed at about 50 °C in 6 x SSC, and a high stringency hybridization reaction is generally performed at about 60 °C in 1 x SSC. Hybridization reactions can also be performed under "physiological conditions" which is well known to one of skill in the art. A non-limiting example of a physiological condition is the temperature, ionic strength, pH and concentration of Mg²⁺ normally found in a cell.

[0077] When hybridization occurs in an antiparallel configuration between two

single-stranded polynucleotides, the reaction is called "annealing" and those polynucleotides are described as "complementary". A double-stranded polynucleotide can be

"complementary" or "homologous" to another polynucleotide, if hybridization can occur between one of the strands of the first polynucleotide and the second. "Complementarity" or "homology" (the degree that one polynucleotide is complementary with another) is quantifiable in terms of the proportion of bases in opposing strands that are expected to form hydrogen bonding with each other, according to generally accepted base-pairing rules. [0078] CD 105 is also known as Endoglin (ENG) is reported to be a 658 amino acid sequence and a homodimer that forms a heteromeric complex with the signaling receptors for transforming growth factor-beta (TGFBR). A polynucleotide encoding the marker and an amino acid sequence is disclosed under GenBank No. M_001278138 (see also

genecards. org/cgi-bin/carddisp.pl?gene=ENG, last accessed on August 13, 2014). Antibodies to the marker are commercially available from numerous vendors, e.g., R&D Systems Antibodies, Novus Biologicals and Abeam antibodies.

[0079] CD90 also is known as Thy-1. A polynucleotide encoding the marker and an amino acid sequence are disclosed under GenBank number NM_006288. Additional information regarding the marker and vendors that provide antibodies to the marker are disclosed under Genecards reference: genecards.org/cgi-bin/carddisp.pl?gene=THYl , last accessed on August 13, 2014.

[0080] CD73 also is known as NT5E. The protein is reported to be a gene is a plasma membrane protein that catalyzes the conversion of extracellular nucleotides to membrane- permeable nucleosides. The encoded protein is used as a determinant of lymphocyte differentiation. Defects in this gene can lead to the calcification of joints and arteries. Two transcript variants encoding different isoforms have been reported for this gene. See genecards.org/cgi-bin/carddisp.pl?gene=NT5E , last accessed on August 13, 2014. A polynucleotides encoding the protein and an encoded amino acid sequences are disclosed under GenBank number BC065937. Antibodies to the marker are commercially available from several vendors, e.g., R&D Systems Antibodies.

[0081] CD44 is reported to be a cell-surface glycoprotein involved in cell-cell interactions, cell adhesion and migration. It is a receptor for hyaluronic acid (HA) and can also interact with other ligands, such as osteopontin, collagens, and matrix metailoproteinases (MMPs). A polynucleotide and encoded amino acid sequence are disclosed under GenBank number FJ216964 (last accessed August 13, 2014). Additional information regarding the marker and commercially available antibodies to the marker are disclosed at genecards.org/cgi- bin/carddisp.pl?gene=CD44, last accessed on August 13, 2014.

[0082] CD29 also is known as Integrin Beta 1 , Fibronectin Receptor, Beta Polypeptide (see Genecards: genecards.org/cgi-bin/carddisp.pl?gene=ITGBl , last accessed on August 13, 2014). A polynucleotide and protein encoded by the polynucleotide are disclosed under GenBank number NG_029012. Additional information regarding the marker and commercially available antibodies to the marker are disclosed at genecards.org/cgi- bin/carddisp.pl?gene=CD44, last accessed on August 13, 2014.

[0083] CD31 also is known as platelet/endothelial cell adhesion molecule 1 (PECAM1). A polynucleotide and amino acid sequence encoded by it is reported under GenBank number AF281301. Additional information regarding the marker and commercially available antibodies to the marker are disclosed at genecards.org/cgi-bin/carddisp.pl?gene=PECAMl , last accessed on August 13, 2014.

[0084] CD34 is a cell surface marker. A polynucleotide and amino acid sequence encoded by it is reported under GenBank number M81 104 (X60172). Additional information regarding the marker and commercially available antibodies to the marker are disclosed at genecards.org/cgi-bin/carddisp.pl?gene=CD34, last accessed on August 13, 2014.

[0085] CD45 also is known as protein tyrosine phosphatase, receptor type C (PTPRC). A polynucleotide and amino acid sequence encoded by it is reported under GenBank number AY538691. Additional information regarding the marker and commercially available antibodies to the marker are disclosed at genecards.org/cgi-bin/carddisp. pl?gene=PTPRC, last accessed on August 13, 2014.

[0086] A "viral vector" is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors and the like. Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. See, Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 and Ying, et al. (1999) Nat. Med. 5(7):823-827.

[0087] In aspects where gene transfer is mediated by a lentiviral vector, a vector construct refers to the polynucleotide comprising the lentiviral genome or part thereof, and a therapeutic gene. As used herein, "lentiviral mediated gene transfer" or "lentiviral transduction" carries the same meaning and refers to the process by which a gene or nucleic acid sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome. The virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell. Retroviruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus. As used herein, lentiviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism. A "lentiviral vector" is a type of retroviral vector well-known in the art that has certain advantages in transducing nondividing cells as compared to other retroviral vectors. See, Trono D. (2002) Lentiviral vectors, New York: Spring- Verlag Berlin Heidelberg.

[0088] Lentiviral vectors of this invention are based on or derived from oncoretroviruses (the sub-group of retroviruses containing MLV), and lentiviruses (the sub-group of retroviruses containing HIV). Examples include ASLV, SNV and RSV all of which have been split into packaging and vector components for lentiviral vector particle production systems. The lentiviral vector particle according to the invention may be based on a genetically or otherwise (e.g., by specific choice of packaging cell system) altered version of a particular retrovirus.

[0089] That the vector particle according to the invention is "based on" a particular retrovirus means that the vector is derived from that particular retrovirus. The genome of the vector particle comprises components from that retrovirus as a backbone. The vector particle contains essential vector components compatible with the R A genome, including reverse transcription and integration systems. Usually these will include gag and pol proteins derived from the particular retrovirus. Thus, the majority of the structural components of the vector particle will normally be derived from that retrovirus, although they may have been altered genetically or otherwise so as to provide desired useful properties. However, certain structural components and in particular the env proteins, may originate from a different virus. The vector host range and cell types infected or transduced can be altered by using different env genes in the vector particle production system to give the vector particle a different specificity.

[0090] As used herein, a "pluripotent cell" also termed a "stem cell" defines a less differentiated cell that can give rise to at least two distinct (genotypically and/or

phenotypically) further differentiated progeny cells. In another aspect, a "pluripotent cell" includes a Induced Pluripotent Stem Cell (iPSC) which is an artificially derived stem cell from a non-pluripotent cell, typically an adult somatic cell, produced by inducing expression of one or more stem cell specific genes. Such stem cell specific genes include, but are not limited to, the family of octamer transcription factors, i.e., Oct-3/4; the family of Sox genes, i.e., Soxl , Sox2, Sox3, Sox 15 and Sox 18; the family of Klf genes, i.e., Klfl , Klf2, Klf4 and Klf5; the family of Myc genes, i.e. c-myc and L-myc; the family of Nanog genes, i.e., OCT4, NANOG and REX1 ; or LIN28. Examples of iPSCs are described in Takahashi et al. (2007) Cell advance online publication 20 November 2007; Takahashi & Yamanaka (2006) Cell 126:663-76; Okita et al. (2007) Nature 448:260-262; Yu et al. (2007) Science advance online publication 20 November 2007; and Nakagawa et al. (2007) Nat. Biotechnol. Advance online publication 30 November 2007.

[0091] As used herein, a "pluripotent cell" defines a less differentiated cell that can give rise to at least two distinct (genotypically and/or phenotypically) further differentiated progeny cells.

[0092] A "multi-lineage stem cell" or "multipotent stem cell" refers to a stem cell that reproduces itself and at least two further differentiated progeny cells from distinct developmental lineages. The lineages can be from the same germ layer (i.e., mesoderm, ectoderm or endoderm), or from different germ layers. An example of two progeny cells with distinct developmental lineages from differentiation of a multilineage stem cell is a myogenic cell and an adipogenic cell (both are of mesodermal origin, yet give rise to different tissues). Another example is a neurogenic cell (of ectodermal origin) and adipogenic cell (of mesodermal origin).

[0093] As used herein, "stem cell" defines a cell with the ability to divide for indefinite periods in culture and give rise to specialized cells. At this time and for convenience, stem cells are categorized as somatic (adult) or embryonic. A somatic stem cell is an

undifferentiated cell found in a differentiated tissue that can renew itself (clonal) and (with certain limitations) differentiate to yield all the specialized cell types of the tissue from which it originated. An embryonic stem cell is a primitive (undifferentiated) cell from the embryo that has the potential to become a wide variety of specialized cell types. An embryonic stem cell is one that has been cultured under in vitro conditions that allow proliferation without differentiation for months to years. A clone is a line of cells that is genetically identical to the originating cell; in this case, a stem cell.

[0094] The term "an expression control element" as used herein, intends a polynucleotide that is operatively linked to a target polynucleotide to be transcribed, and facilitates the expression of the target polynucleotide. A promoter is an example of an expression control element. [0095] A promoter is a regulatory polynucleotide, usually located 5 ' or upstream of a gene or other polynucleotide, that provides a control point for regulated gene transcription.

Polymerase II and III are examples of promoters.

[0096] A polymerase II or "pol Π" promoter catalyzes the transcription of DNA to synthesize precursors of mRNA, and most shKNA and microR A. Examples of pol II promoters are known in the art and include without limitation, the phosphoglycerate kinase ("PG ") promoter; EF 1 -alpha; CMV (minimal cytomegalovirus promoter); MNDU3 ; and LTRs from retroviral and lentiviral vectors. A non-limiting example is provided from nucleotides 4661 to 5204 of SEQ ID NOs. 1 , 2 or 5.

[0097] A polymerase III or "pol ΙΠ" promoter is a polynucleotide found in eukaryotic cells that transcribes DNA to synthesize ribosomal 5S rRNA, tRNA and other small RNAs. An examples of pol III promoters include without limitation a U6 promoter.

[0098] A "target cell" as used herein, shall intend a cell containing the genome into which polynucleotides that are operatively linked to an expression control element are to be integrated.

[0099] As used herein and known to the skilled artisan, a "marker" is a receptor or protein expressed by the cell or internal to the cell which can be used as an identifying and/or distinguishing factor. If the marker is noted as ("+"), the marker is positively expressed. If the marker is noted as ("-"), the marker is absent or not expressed. Variable expression of markers are also used, such as "high" and "low" and relative terms.

[0100] As used herein, the term "detectable marker" intends a polynucleotide, detectable label or other molecule that allows for the identification of a preselected composition. Non- limiting examples of reporter markers include, without limitation a hemmaglutinin tag, an enhanced green fluorescent protein (EGFP), a red flouresence protein (RFP), a green fluorescent protein (GFP) and yellow fluorescent protein (YFP), YUC or the like. These are commercially available and described in the technical art.

[0101] The terms effective period (or time) and effective conditions refer to a period of time or other controllable conditions (e.g., temperature, humidity for in vitro methods), necessary or preferred for an agent or composition to achieve its intended result, e.g., the differentiation of cells to a pre-determined cell type.

[0102] A "composition" is also intended to encompass a combination of active agent and another carrier, e.g., compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like. Carriers also include biocompatible scaffolds, pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume. Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid/antibody

components, which can also function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. Carbohydrate excipients are also intended within the scope of this invention, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like;

polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.

[0103] The term pharmaceutically acceptable carrier (or medium), which may be used interchangeably with the term biologically compatible carrier or medium, refers to reagents, cells, compounds, materials, compositions, and/or dosage forms that are not only compatible with the cells and other agents to be administered therapeutically, but also are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other complication commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable carriers suitable for use in the present invention include liquids, semi-solid (e.g., gels) and solid materials (e.g., cell scaffolds and matrices, tubes sheets and other such materials as known in the art and described in greater detail herein). These semi-solid and solid materials may be designed to resist degradation within the body (non-biodegradable) or they may be designed to degrade within the body (biodegradable, bioerodable). A biodegradable material may further be bioresorbable or bioabsorbable, i.e., it may be dissolved and absorbed into bodily fluids (water-soluble implants are one example), or degraded and ultimately eliminated from the body, either by conversion into other materials or breakdown and elimination through natural pathways.

[0104] A "control" is an alternative subject or sample used in an experiment for comparison purpose. A control can be "positive" or "negative". For example, where the purpose of the experiment is to determine a correlation of an altered expression level of a gene with a particular phenotype, it is generally preferable to use a positive control (a sample from a subject, carrying such alteration and exhibiting the desired phenotype), and a negative control (a subject or a sample from a subject lacking the altered expression or phenotype).

Additionally, when the purpose of the experiment is to determine if an agent effects the differentiation of a stem cell, it is preferable to use a positive control (a sample with an aspect that is known to affect differentiation) and a negative control (an agent known to not have an affect or a sample with no agent added).

[01051 The terms autologous transfer, autologous transplantation, autograft and the like refer to treatments wherein the cell donor is also the recipient of the cell replacement therapy. The terms allogeneic transfer, allogeneic transplantation, allograft and the like refer to treatments wherein the cell donor is of the same species as the recipient of the cell replacement therapy, but is not the same individual. A cell transfer in which the donor's cells and have been histocompatibly matched with a recipient is sometimes referred to as a syngeneic transfer. The terms xenogeneic transfer, xenogeneic transplantation, xenograft and the like refer to treatments wherein the cell donor is of a different species than the recipient of the cell replacement therapy.

[0106] A population of cells intends a collection of more than one cell that is identical (clonal) or non-identical in phenotype and/or genotype.

[0107] "Substantially homogeneous" describes a population of cells in which more than about 50%, or alternatively more than about 60 %, or alternatively more than 70 %>, or alternatively more than 75 %, or alternatively more than 80%, or alternatively more than 85 %, or alternatively more than 90%, or alternatively, more than 95 %, of the cells are of the same or similar phenotype. Phenotype can be determined by a pre-selected cell surface marker or other marker. A clonal population is a population that is expanded or derived from a single cell or one that is at least 98 %, or alternatively at least 99%, or alternatively about 100%) identical phenotype. [0108] A "subject," "individual" or "patient" is used interchangeably herein, and refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, rats, rabbit, simians, bovines, ovine, porcine, canines, feline, farm animals, sport animals, pets, equine, and primate, particularly human. Besides being useful for human treatment, the present invention is also useful for veterinary treatment of companion mammals, exotic animals and domesticated animals, including mammals, rodents, and the like which is susceptible to Hemophilia A. In one embodiment, the mammals include horses, dogs, and cats. In another embodiment of the present invention, the human is an adolescent, an infant under the age of eighteen years of age or a fetus.

[0109] "Host cell" refers not only to the particular subject cell but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0110] An "enriched population" of cells intends a substantially homogenous population of cells having certain defined characteristics. The cells are greater than 70 %, or alternatively greater than 75 %, or alternatively greater than 80 %, or alternatively greater than 85 %, or alternatively greater than 90 %, or alternatively greater than 95 %, or alternatively greater than 98% identical in the defined characteristics.

[0111] "Treating" or "treatment" of a disease includes: (1 ) preventing the disease, i.e., causing the clinical symptoms of the disease not to develop in a patient that may be predisposed to the disease but does not yet experience or display symptoms of the disease; (2) inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms; (3) relieving the disease, i.e., causing regression of the disease or its clinical symptoms; (4) expression of functional protein in the patient having received treatment.

[0112] The term "suffering" as it related to the term "treatment" refers to a patient or individual who has been diagnosed with or is predisposed to Hemophilia A. A patient may also be referred to being "at risk of suffering" from a disease because the presence of an allele defective in Factor VIII production and has not yet developed characteristic disease pathology, such as a fetus.

[0113] An "effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents of the present invention for any particular subject depends upon a variety of factors including the activity of the specific agent employed, the age, body weight, general health, sex, and diet of the subject, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of

administration. Treatment dosages generally may be titrated to optimize safety and efficacy. Typically, dosage-effect relationships from in vitro and/or in vivo tests initially can provide useful guidance on the proper doses for patient administration. In general, one will desire to administer an amount of the compound that is effective to achieve a serum level

commensurate with the concentrations found to be effective in vitro. Determination of these parameters is well within the skill of the art. These considerations, as well as effective formulations and administration procedures are well known in the art and are described in standard textbooks.

[0114] The term administration shall include without limitation, in utero

administrationadministration by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection, or implant), by inhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository) or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.) and can be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration. In one aspect, the method for administration of the therapeutic cells comprises in utero delivery to the festus, e.g., by percutaneous, ultrasound guided, intraperitoneal injection, at various times during gestational development, e.g., 16 weeks', 17. 5 weeks', and 18.5 weeks' gestation. In addition, it is possible to deliver the therapeutic cells after 18 weeks' gestation by funneling cells through the ubmiliical vein. See, e.g., Flacke et al. ( 1996) N. Engl. J. Med. Vol. 335 : 1806- 1810 and Wengler, et al. (1996) Lancet, Vol. 348: 1484- 1487. The invention is not limited by the route of administration, the formulation or dosing schedule.

[0115] Methods for transplantation of cells to a fetus in utero have been described in the literature, e.g., Vrecenak et al. (2013) In utero hematopoietic cell transplantation-recent progress and the potential for clinical application, Cytotherapy, Vol. 15(5):525-35; Pearson, et al. (2010) Stem cell and genetic therapies for the fetus, Semin Pediatr Surg Vol. 22(1 ): 56- 61 ; and Nijagal A, et al. (2013) Clinical implications of maternal-fetal cellular trafficking, Semin Pediatr Surg. Vol. 22( l):62-65.

Descriptive Embodiments

[0116] This disclosure provides methods to treat or prevent genetic diseases or disorders, for example Hemophilia A, a lysosomal storage disorder (LSD) or Duchenne's Muscular Dystrophy (DMD), in a subject in need thereof, comprising, or alternatively consisting essentially of, or yet further consisting of, administering to the subject an effective amount of a nucleic acid encoding a therapeutic, functional polypeptide, e.g., a Factor VIII polypeptide for the treatment of Hemophilia A or dystrophin for the treatment of DMD. In one aspect, the disease to be treated is Hemophilia A and the protein is Factor VIII polypeptides. Non- limiting examples of Factor VIII polypeptides include wildtype Factor VIII, a fragment thereof, a B-domain deleted Factor VIII polypeptide (FVIII) or an equivalent thereof, a chimeric polypeptide encoding functional Willebrand factor polypeptide, or a fusion polypeptide, or an equivalent thereof. The nucleic acid is administered in a cell isolated from placenta, such as a pre-term placenta-derived stem cell (also referred to chorionic villus sampling tissue derived stem cells ("CSCs") isolated from the mother carrying the fetus. Non-limiting examples of cells to transfer the nucleic acid include mesenchymal stem cells (C-MSCs), hemaptoieitc stem cells (C-HSCs), epithelial progenitor cells, or endothelial progenitor cells that also can be isolated from this tissue. Examples also include other cells, such as endothelial cells, that can be derived or differentiated from these pre-term placenta- derived stem cells. As used herein, the term "pre-term placenta-derived stem cell" intends a cell isolated from placental tissue prior to delivery of the fetus by surgery or birth. In another aspect, the isolated cell is a pre-term chorionic villus tissue derived stem cell (CSC) comprising the nucleic acid encoding the functional Factor VIII polypeptide. In an alternate embodiment, the isolated cell further comprises a nucleic acid encoding functional von Willebrand factor. In one aspect, the cells are administered in utero to the festus, e.g., by percutaneous, ultrasound guided, intraperitoneal injection, at various times during gestational development, e.g., 1 6 weeks', 17. 5 weeks', and 1 8.5 weeks' gestation. In addition, it is possible to deliver the therapeutic cells after 1 8 weeks' gestation by funneling cells through the ubmiliical vein.

[0117] The patient or subject is treated by expression of the functional protein in the subject in, or by a cell in subject in need of such treatment. For Hemophilia, one non-limiting measure of treatment is the expression of functional Factor VIII polypetide by the patient. For DMD, one non-limitng measure of treatment is the production of dystrophin in a cell or tissue in the patient having been treated. For LSDs, a non-limiting meaure of treatment is expression of the enzyme whose functional absence gives rise to clinical symptoms. Methods to determine if functional protein is being expressed are known in the art, e.g., by the use of , polymerase chain reaction, histology and the use antibodies that bind functional protein. In addition, and after birth of the fetus, the child can be monitored for clinical symptoms of the genetic disorder. A reduction or complete elimination of clinical symptons are alternative indications of effective therapy.

[0118] The method of this disclosure is not limited by which the nucleic acid encoding the therapeutic protein is inserted into the cell, eg.a., a pre-term placenta-derived cell. Non- limited examples include vector-mediated insertion, CRISPR (CRISPR-Cas9) or TALENs.

[0119] In one aspect, the nucleic acid encoding functional Factor VIII polypeptide and/or the nucleic acid encoding funcational von Willebrand factor is contained within a vector that is used to transduce the cell with the nucleic acid(s). Non-limiting examples of such polypeptides include wildtype von Willebrand factor, a fragment thereof, a chimeric polypeptide encoding functional Willebrand factor polypeptide, or a fusion polypeptide, or an equivalent thereof. The vector comprising the nucleic acid encoding functional Factor VIII polypepide (see, e.g., SEQ ID NO. 1 , from about nucleotide 5235 to about 9742), can in one embodiment, further comprise a polynucleotide encoding functional von Willebrand factor. The vectors can further comprise a promoter operatively linked to the nucleic acids, non- limiting examples of such are provided in the Sequence Listing attached hereto and incorporated herein by reference. In embodiments wherein a single vector comprise a nucleic acid encoding the functional Factor VIII polypeptide and a nucleic acid encoding functional von Willebrand factor, each is operatively linked to a promoter. Non-linking examples of promoters to drive expression of the nucleic acids include an MDNU3 promoter (SEQ ID NO. 5, nucleotide about 4661 to about 4652, or an equivalent thereof) or a PGK promoter (SEQ ID NO.: 5, from about nucleotide 9721 to about 10236, or an equivalent thereof). In an another aspect, the vector further comprises one or more enhancer elements, a non-limiting example of such is disclosed in SEQ ID NO. 2, from about nucleotide 9757 to about 10353, or an equivalent thereof. In a further aspect, the polynucleotide encoding functional von Willebrand factor can be in a separate vector and transduced into the same cell or an another isolated cell for co-administration to the subject in need of this therapy. Modificiations of this described method are made by substitution of a nucleic acid encoding the therapeutic, functional protein appropriate for the genetic disorder, e.g., a nucleic acid encoding functional dystrophin protein for the treatment of DMD.

[0120] In one aspect, the compositions and methods described herein are useful in the treatment of a fetus, such as a human fetus. In one aspect, the cells to be transduced with the vectors described herein are first isolated from the early gestational placenta, in one aspect, the mother carrying the fetus to be treated. The cells are then transduced with the vector comprising the nucleic acid encoding functional Factor VIII alone or in combination with a nucleic acid encoding functional von Willebrand factor. In one aspect, the isolated, transduced cells are expanded to a population of cells containing the nucleic acids prior administration to the subject, e.g., the fetus. The therapeutic methods as described herein can be combined with a pre-term diagnostic to determine if the fetus is likely to develop the genetic disease or disorder, e.g., Hemophilia A, prior to the administration of the therapy. Such methods are known in the art. Methods to detect genetic disorder are generally described at the web addresss: genome.gov/10001204/specific-genetic-disorders/, last accessed on May 14, 2016.

[0121] This disclosure also provides the compositions to prepare the cells for the therapy. In one aspect, a vector is provided that is used to transduce the isolated cells used for the therapy, such as an isolated CSC. The vector comprises: (a) a backbone comprising essential sequences for integration into a target cell genome; (b) a nucleic acid encoding a functional Factor VIII polypeptide; and (c) a first expression control element that regulates expression of the nucleic acid encoding the functional Factor VIII polypeptide. In another aspect, the vector further comprises (d) a nucleic acid encoding a marker polypeptide and (e) a second control element that regulates expresson of the nucleic acid encoding the marker polypeptide. In a further aspect, the vector futher comprises (e) a nucleic acid encoding functional von Willebrand factor and (f) a second control element that regulates expression of the functional von Willebrand factor. In a yet further aspect, the vector further comprises (g) one or more enhancer elements. In a yet further aspect, the vector further comprises (h) a P2A an internal protease coding site (a non-limiting example of such is from about nucleotide 1 1909 to about 1 1974 of SEQ ID NO. 5), wherein P2A is the nucleotide sequence encoding a peptide that promotes a ribosomal skip resulting in the stoichiometric expression of two unfused reporter proteins from the same mRNA transcript. In further aspects, the disclosure provides vector comprises: (a) a backbone comprising essential sequences for integration into a target cell genome; (b) a nucleic acid encoding a functional von Willebrand factor; and (c) an expression control element that regulates expression of the nucleic acid encoding the functional von Willebrand factor. The vector can further comprise a nucleic acid encoding a marker polypeptide under the control of a promoter element. The vector can further comprise an enhancer element. In one aspect the vectors are viral vectors, such as lentiviral vectors.

[0122] In one embodiment, the one or more expression control elements are Polymerase II promoters, non-limiting examples of such are MNDU3 and phosphoglycerate kinase (PGK) promoter. Non-limiting examples of detectable markers are Green Flourescent Protein (GFP) or luciferase (LUC) A non-limiting example of such are provide in SEQ ID NO. 5 from about nucleotide 10259 to about 1 1908 (LUC) and from about nucleotide 1 1975 to about 12694 (GFR) . A non-limiting example of an enhancer element is WPRE. A non-limiting example of a vector comprising these elements is provided in SEQ ID NOs: 1 , 2 or 5 or a

polynucleotide having at least 80 % identity thereto. The disclosed vectors can be modified for expression of other suitable therapeutic polynucleotides or further modified to expressly omit the maker elements, e.g., the vectors as disclosed in SEQ ID NOs. 1 , 2 and 5 absent the sequences and regulatory elements for the detectable labels, e.g., GFP and/or LUC.

[0123] This disclosure also provides a viral packaging system comprising: (a) the vector(s) as described above wherein the backbone is derived from a virus; (b) a packaging plasmid; and (c) an envelope plasmid. In one aspect, the envelope plasmid is a plasmid comprising a S. aureus ZZ domain sequence, or a sequence encoding a pseudotyped envelope protein or a VSVG envelope, or a pMD.G VSV sequence or other envelope plasmids known in the art and available from addgene (addgene.org/12259, last accessed on May 12, 2015). Also provided herein is a packaging cell line such as HEK-293 cell for viral production.

[0124] Further provided herein is a method for producing a pseudotyped viral particle that expresses a functional Factor VIII polypeptide and/or functional von Willebrand factor by transducing one or more packaging cell lines as described above with the system as described above under conditions suitable to package the viral vector, as well as the pseudotyped viral particle produced by the methods.

[0125] As noted above, the pseudotyped viral particle is useful to transduce an isolated cell such as a CSC cell which in turn is useful for the treatment or prevention of Hemophilia A in a subject in need thereof. Thus, this disclosure also provides the pseudotyped viral particle conjugated or attached to the isolated cell to be infected by the virus. Accordingly, also provided is the isolated cell comprising one or more of: the nucleic acids and/or the vectors and/or the pseudotyped viral particles as described herein.

[0126] Also as noted above, the compositions are useful in a therapeutic method to treat a fetus determined to be at risk of developing Hemophilia A upon birth and the nucleic acid encoding functional Factor VIII is contained within the cell, such as a stem cell isolated from pre-term placental tissue. Non-limiting example of such cells include mesenchymal stem cells (C-MSCs), hemaptoieitc stem cells (C-HSCs), epithelial progenitor cells, and endothelial progenitor cells. Applicants have determined that C-MSCs that are useful in the method express one or more of the markers CDl 05, CD90, CD73, CD44, and CD29 and did not express one or both of CDl 84 and/or HLA-DR. In addition or alternatively, the C-MSCs do not express the markers CD45, CD34 and/or CD31 and in one aspect, are isolated from early gestation placenta.

[0127] Also provided is a population of C-MSCs as described herein. Such population can be a substantially homogenous population of cells such as a clonal population. Alternatively, the stem cells can be expanded and differentiated into a population of one or more mesodermal lineages, e.g. osteogenic cell, an adipogenic cell or a chondrogenic cell.

[0128] Any one or more of the cells, vectors, packaging systems, and/or pseudotyped viral particles can be combined with a carrier, such as a pharmaceutically acceptable carrier.

[0129] Yet further provided is a method to express a functional FactorVIII polypeptide by culturing the cell as described above under conditions that favor expression of the functional FactorVIII polypeptide. In a further aspect, the same cell or an additional cell comprises a vector further comprises a polynucleotide encoding functional von Willebrand factor under the control of the same first promoter or its own Polymerase II promoter, examples of such are known in the art and described herein. In a further aspect, the polynucleotide encoding von Willebrand factor can be in a separate vector and transduced into the same cell or an another isolated cell for co-administration to the subject in need of this therapy. In a further aspect, the method further comprises isolating the functional Factor VIII polypeptide and/or von Willebrand factor prepared by the method.

[0130] Yet further provided is a method for form ing a matrix, comprising, or alternatively consisting essentially of, or yet further consisting of, combining a population of cells as described above with a pharmaceutically acceptable carrier such as a biocompatible matrix or scaffold, optionally suitable for implantation in vivo e.g., in utero. [0131] Kits are also provided, having the compositions described above, alone or in combination, and optionally, reagents and instructions for use of one or more of:

diagnostically, as a research tool or therapeutically.

Modes For Carrying Out the Disclosure

[0132] Applicants have determined that the placenta, a unique organ containing tissue autologous to the fetus, is an ideal cell source for in utero stem cell therapy. In one aspect, placental tissue can be obtained via chorionic villous sampling (CVS), an established diagnostic technique (Ahmed, S. (2006) J. Coll. Physicians Surg. Pak. 16:204-207). Unlike allogeneic cells, autologous cells carry no risk of triggering an adverse immune reaction. Most fetal autologous cell sources can only be accessed at considerable risk to the fetus or provide cells difficult to expand on a prenatal timeline (Kunisaki, S.M. et al. (2006) J.

Pediatr. Surg. 41 :675-682; Golombeck, K. et al. (2006) Am. J. Obstet. Gynecol. 194:834- 839). In contrast, the placenta can be accessed via CVS relatively safely early in the pregnancy (Alfirevic, Z. et al. (2009) The Cochrane Library 1 : 1- 143). Using CVS-sized samples of preterm placenta, Applicants developed a protocol to isolate CVS-derived multipotent placental stem cells, characterized the cells and determined that they are a variant of mesenchymal stem cell (MSC) that can differentiate into mesodermal (osteogenic, adipogenic, chondrogenic) lineages. These cells can be used for autologous or allogeneic therapy.

[0133] The cells are genetically modified to express a therapeutic functional protein, e.g., functional dystrophin, N-sulfoglucosamine sulfohydrolase (SGSH) gene, factor VIII, e.g., human Factor VIII (hFVIII) using any appropriate transduction method, e.g., CRISPR, TALENs, or vector-mediated transfer, e.g., a viral vector and transplanted into a mammal. In utero xenogeneic transplantation of the gene modified human C-MSCs that express a functional, therapeutic protein, e.g.a, hFVIII. C-MSCs isolated from preterm human placenta (GA 10-12 weeks) are plastic adherent, showed spindle-shaped morphology and

demonstrated expression of mesenchymal stem cell markers CD105, CD90, CD73, CD44, and CD29, and did not express CD184, HLA-DR or hematopoietic and endothelial markers CD45, CD34 and CD31. In addition or alternatively, the cells express the markers CD45, CD34 and/or CD31 and in one aspect, are isolated from early gestation placenta. Tri-lineage differentiation potential into osteogenic, adipogenic and chondrogenic lineages was observed under different conditions. These results show multi-potency and a surface marker profile analogous to bone marrow mesenchymal stem cells (BMSCs). [0134] In one aspect when Hemophilia A is to be treated, the cells are genetically modified to express functional factor VIII, e.g., human Factor VIII (hFVIII) using a viral vector and transplanted into a mammal. In utero xenogeneic transplantation of the gene modified human C-MSCs that express hFVIII. C-MSCs isolated from preterm human placenta (GA 10-12 weeks) were plastic adherent, showed spindle-shaped morphology and demonstrated expression of mesenchymal stem cell markers CD 105, CD90, CD73, CD44, and CD29, and did not express CD184, HLA-DR or hematopoietic and endothelial markers CD45, CD34 and CD3 1 . In addition or alternatively, the cells express the markers CD45, CD34 and/or CD3 1 and in one aspect, are isolated from early gestation placenta. Tri-lineage differentiation potential into osteogenic, adipogenic and chondrogenic lineages was observed under different conditions. These results show multi-potency and a surface marker profile analogous to bone marrow mesenchymal stem cells (BMSCs).

[0135] In one aspect, the C-MSCs are transduced for FVIII transgene expression via lentiviral vector with B-domain deleted hFVIII ("F8"), GFP and luciferase (LUC), and driven by MNDU promoter (pCCLc-M DU3-EGFP/LUC-PGK-F8-WPRE). At an MOI

(Multiplicity of infection) of 3, lentiviral vector transduction resulted in GFP expression of 90-95% of C-MSCs, assessed via immunofluorescence and flow cytometry, without alteration in morphology or proliferative capacity. Immunocytochemistry, using an antibody specific to hFVIII and HUVECs as negative control, showed that transduced C-MSCs expressed hFVIII. Furthermore, functional hFVIII was detected in cell supernatant by chromogenic assay: 4.5 IU/10 cells (-150% factor activity / 10 cells) and FVIII protein was measured using an enzymelinked immunosorbent assay (ELISA): 24 ng/10 cells. C-MSCs were transplanted intraperitoneally into murine fetuses (embryonic age 12.5 days) with a 33 gauge non-coring needle. Bioluminescence imaging analysis revealed focal density and transgene expression in the fetuses in utero 1 -3 days after transplantation and subsequently in the postnatal pups 12- 1 5 days after transplantation. This data demonstrate that intrauterine transplantation of gene modified human C-MSCs via a lentiviral vector can be a reasonable approach for cell therapy, and further suggest the potential of in utero approaches for tolerance induction. Additionally, the data showed sustained hFVIII transgene expression with cell survivability and safety in the transplanted mice, suggesting that C-MSCs are a stem cell source for the prenatal phenotypic correction of Hemophilia A. Vectors, Packaging Cell Lines Encoding Factor VIII and von Willebrand Factor Factor VIII and von Willebrand Factor

[0136] The disclosure provides a vector comprising a polynucleotide encoding a functional Factor VIII, (non-limiting examples of such include wild-type Factor VIII, B-domain deleted Factor VIII polypeptide (hFVIII), a fragment thereof, a fusion thereof, a chimeric polypeptide encoding functional Factor VIII polypeptide, or an equivalent of each thereo) that phenotypically corrects Hemophilia A. In one aspect the vector further comprises a polynucleotide encoding functional von Willebrand factor (non-limiting examples of such include wild-type von Willebrand factor, a fragment thereof, a fusion thereof, a chimeric polypeptide encoding functional von Willebrand factor polypeptide or an equivalent of each thereof. Polynucleotides that encode such functional Factor VIII (e.g., wild-type, a fragment or a chimeric polypeptide encoding functional Factor VIII polypeptide) and functional von Willebrand factor (e.g., a fragment or a chimeric polypeptide encoding functional von Willebrand factor polypeptide) are known in the art, and described in U.S. Patent Application Publication Nos. 2014/0357564; 2014/0072561 ; 2014/0056861 ; 2013/0017997 (Factor VIII polypeptide linked to (extended recombinant polypeptide (XTEN)); 2013/0296244,

2013/0108629 (Factor VIII-Fc chimeric and hybrid polypeptides); and 2013/0267468.

Viral Vectors

[0137] In one aspect, the term "vector" intends a recombinant vector that retains the ability to infect and transduce non-dividing and/or slowly-dividing cells and integrate into the target cell's genome. In several aspects, the vector is derived from or based on a wild-type virus. In further aspects, the vector is derived from or based on a wild-type lentivirus. Examples of such, include without limitation, human immunodeficiency virus (HIV), equine infectious anaemia virus (EIAV), simian immunodeficiency virus (SIV) and feline immunodeficiency virus (FIV). Alternatively, it is contemplated that other retrovirus can be used as a basis for a vector backbone such murine leukemia virus (MLV). It will be evident that a viral vector according to the invention need not be confined to the components of a particular virus. The viral vector may comprise components derived from two or more different viruses, and may also comprise synthetic components. Vector components can be manipulated to obtain desired characteristics, such as target cell specificity.

[0138] The recombinant vectors of this invention are derived from primates and non- primates. Examples of primate lentiviruses include the human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS), and the simian immunodeficiency virus (SIV). The non-primate lentiviral group includes the prototype "slow virus" visna/maedi virus (VMV), as well as the related caprine arthritis- encephalitis virus (CAEV), equine infectious anaemia virus (EIAV) and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV). Prior art recombinant lentiviral vectors are known in the art, e.g., see U.S. Patent Nos.

6,924, 123; 7,056,699; 7,07,993; 7,419,829 and 7,442,551 , incorporated herein by reference.

[0139] U.S. Patent No. 6,924,123 discloses that certain retroviral sequence facilitate integration into the target cell genome. This patent teaches that each retroviral genome comprises genes called gag, pol and env which code for virion proteins and enzymes. These genes are flanked at both ends by regions called long terminal repeats (LTRs). The LTRs are responsible for proviral integration, and transcription. They also serve as enhancer-promoter sequences. In other words, the LTRs can control the expression of the viral genes.

Encapsidation of the retroviral RNAs occurs by virtue of a psi sequence located at the 5' end of the viral genome. The LTRs themselves are identical sequences that can be divided into three elements, which are called U3, R and U5. U3 is derived from the sequence unique to the 3' end of the RNA. R is derived from a sequence repeated at both ends of the RNA, and U5 is derived from the sequence unique to the 5'end of the RNA. The sizes of the three elements can vary considerably among different retroviruses. For the viral genome, and the site of poly (A) addition (termination) is at the boundary between R and U5 in the right hand side LTR. U3 contains most of the transcriptional control elements of the provirus, which include the promoter and multiple enhancer sequences responsive to cellular and in some cases, viral transcriptional activator proteins. A non-limiting example of a 5' LTR is provided in SEQ ID NOs: 2 from about nucleotide 2358 to about 3072, or an equivalent thereof, and a non-limiting example of a 3 ' LTR is from about nucleotide 10439 to about 10675.

[0140] With regard to the structural genes gag, pol and env themselves, gag encodes the internal structural protein of the virus. Gag protein is proteolytically processed into the mature proteins MA (matrix), CA (capsid) and NC (nucleocapsid). The pol gene encodes the reverse transcriptase (RT), which contains DNA polymerase, associated RNase H and integrase (ΓΝ), which mediate replication of the genome.

[0141] For the production of viral vector particles, the vector RNA genome is expressed from a DNA construct encoding it, in a host cell. The components of the particles not encoded by the vector genome are provided in trans by additional nucleic acid sequences (the "packaging system," which usually includes either or both of the gag/pol and env genes) expressed in the host cell. The set of sequences required for the production of the viral vector particles may be introduced into the host cell by transient transfection, or they may be integrated into the host cell genome, or they may be provided in a mixture of ways. The techniques involved are known to those skilled in the art.

[0142] Retroviral vectors for use in this invention include, but are not limited to

Invitrogen's pLenti series versions 4, 6, and 6.2 "ViraPower" system. Manufactured by Lentigen Corp.; pHIV-7-GFP, lab generated and used by the City of Hope Research Institute; "Lenti-X" lentiviral vector, pLVX, manufactured by Clontech; pLKO. l -puro, manufactured by Sigma-Aldrich; pLemiR, manufactured by Open Biosystems; and pLV, lab generated and used by Charit£ Medical School, Institute of Virology (CBF), Berlin, Germany.

[0143J In other embodiments, the vector further comprise, or alternatively consists essentially of, or yet further consists of a nucleic acid encoding at least functional Factor VIII (e.g., the B-domain deleted human Factor VIII polypeptide (hFVIII)) driven by a Polymerase II promoter (e.g., MNDU3) and optionally a enhancer element, such as WPRE. In a further aspect, the vector further comprises a detectable marker under the control of a Polymerase II promoter, e.g., GFP and/or luciferase, and driven by PGK promoter. The vector can comprise an additional promoter and/or a P2A internal protease coding site, wherein P2A is the nucleotide sequence encoding a peptide that promotes a ribosomal skip resulting in the stoichiometric expression of two unfused reporter proteins from the same mRNA transcript. In one aspect, the vector comprises: a viral backbone, a promoter (MNDU3), a nucleic acid encoding a detectable marker (EGFP/LUC), a promoter (PGK), a nucleic acid encoding functional Factor VIII (B-domain deleted Factor VIII) and an enhancer element (WPRE). In another aspect, the vector comprises a viral backbone, a promoter (MNDU3), a nucleic acid encoding functional Factor VIII polypeptide (B-domain deleted Factor VIII), a promoter element (PGK), a nucleic acid encoding a marker polypeptide (LUC), a-P2A internal protease coding site, wherein P2A is the nucleotide sequence encoding a peptide that promotes a ribosomal skip resulting in the stoichiometric expression of two unfused reporter proteins from the same mRNA transcript, and/or an optional additional enhancer element and a second nucleic acid encoding a detectable marker polypeptide.

[0144] In a further aspect, the vector further comprises a marker or detectable label such as a gene encoding a luciferase (LUC), enhanced green fluorescent protein (EGFP), red flouresence protein (RFP), green fluorescent protein (GFP) and yellow fluorescent protein (YFP) or the like. These are commercially available and described in the technical art.

[0145] In a further aspect, provided herein is a lentiviral vector encoding phenotypically correct Factor VIII, wherein the vector comprises a lentiviral vector backbone operatively linked to the MNDU3 promoter and WPRE enhancer. In a futher aspect, the lentiviral vector backbone contains a polynucleotide encoding a marker such as GFP or LUC linked to a Polymerase II promoter such as the phosphoglycerate kinase (PGK) promoter.

[0146] Examples of vectors for use in this invention are provided in SEQ ID NOs: 1 , 2 or 5. Alternatives include, but are not limited to a polynucleotide having at least 80 % sequence identity, or alternatively at least 85 % sequence identity, or alternatively at least 90 % sequence identity, or alternatively at least 92 % sequence identity, or alternatively at least 95 % sequence identity, or alternatively at least 97 % sequence identity, or alternatively at least 98 % sequence identity to any one of SEQ ID NO: 1 , 2 or 5, or one that hybridizes under stringent conditions to SEQ ID NOs: 1 , 2 or 5 or their complements.

[0147] In a further aspect, the vector further comprises a polynucleotide encoding functional von Willebrand factor (e.g., wild-type protein, a fragment thereof or a chimeric polypeptide encoding functional Factor von Willebrand factor) under the control of the same first promoter or its own Polymerase II promoter, examples of such are known in the art and described herein. In a further aspect, the polynucleotide encoding von Willebrand factor, a fragment thereof or a chimeric polypeptide encoding functional Factor von Willebrand factor can be in a separate vector and transduced into the same cell or a another isolated cell for coadministration to the subject in need of this therapy.

Packaging Systems

[0148] The invention also provides a viral packaging system comprising: the vectors as described above, wherein the backbone is derived from a virus; a packaging plasmid; and an envelope plasmid. The packaging plasmid contains polynucleotides encoding the nucleoside, capsid and matrix proteins. As an example, SEQ ID NO: 3 provides the sequence encoding a packaging plasmid that can be used in this invention. Alternatives include, but are not limited to a polynucleotide having at least 80 % sequence identity, or alternatively at least 85 % sequence identity, or alternatively at least 90 % sequence identity, or alternatively at least 92 % sequence identity, or alternatively at least 95 % sequence identity, or alternatively at least 97 % sequence identity, or alternatively at least 98 % sequence identity to SEQ ID NO: 3 or one that hybridizes under stringent conditions to SEQ ED NO: 3 or its complement. Alternatives are also described in the patent literature, e.g., U.S. Patent Nos. 7,262,049; 6,995,258; 7,252,991 and 5,710,037, incorporated herein by reference.

[0149] The system also contains a plasmid encoding a pseudotyped envelope protein provided by an envelope plasmid. Pseudotyped viral vectors consist of vector particles bearing glycoproteins derived from other enveloped viruses or alternatively constaining functional portions. See, for example U.S. Patent No. 7,262,049, incorporated herein by reference. In a preferred aspect, the envelope plasmid encodes an envelope protein that does not cause the viral particle to unspecifically bind to a cell or population of cells. The specificity of the viral particle is conferred by the antibody binding domain that is inserted into the particle. Examples of suitable envelope proteins include, but are not limited to those containing the Staph, aureus ZZ domain, or the coding sequence provided in SEQ ED NO: 4 or a polynucleotide having at least 80 % sequence identity, or alternatively at least 85 % sequence identity, or alternatively at least 90 % sequence identity, or alternatively at least 92 % sequence identity, or alternatively at least 95 % sequence identity, or alternatively at least 97 % sequence identity, or alternatively at least 98 % sequence identity to that shown in SEQ ED NO: 4 (envelope plasmid pMDG-VSVG), or one that hybridizes under stringent conditions to SEQ ID NO: 4 or its complement, or a sequence encoding a pseudotyped envelope protein or a VSVG envelope, a pMD.G VSV sequence or other envelope plasmids known in the art.

[0150] This invention also provides the suitable packaging cell line. In one aspect, the packaging cell line is the HEK-293 cell line. Other suitable cell lines are known in the art, for example, described in the patent literature within U.S. Patent Nos. 7,070,994; 6,995,919; 6,475,786; 6,372,502; 6,365, 150 and 5,591,624, each incorporated herein by reference.

Pseudotyped Viral Particles

[0151] This invention further provides a method for producing a pseudotyped viral particle, comprising, or alternatively consisting essentially of, or yet further consisting of, transducing a packaging cell line with the viral system as described above, under conditions suitable to package the viral vector. Such conditions are known in the art and briefly described herein. The pseudotyped viral particle can be isolated from the cell supernatant, using methods known to those of skill in the art, e.g., centrifugation. Such isolated particles are further provided by this invention. [0152] This invention further provides the isolated pseudotyped viral particle produced by this method. The pseudotyped viral particle comprises, or alternatively consists essentially of, or yet further consists of a polynucleotide encoding a functional Factor VIII polypeptide, a fragment or a chimeric polypeptide encoding functional Factor VIII polypeptide and envelope protein. In a further aspect, the pseudotyped viral particle further comprises a polynucleotide encoding functional von Willebrand factor, a fragment thereof or a chimeric polypeptide encoding functional von Willebrand polypeptide under the control of the same first promoter or its own Polymerase II promoter, examples of such are known in the art and described herein. In a further aspect, the isolated pseudotyped viral particle produced by this method comprises the polynucleotide encoding von Willebrand factor, a fragment or a chimeric polypeptide encoding functional von Willebrand polypeptide and an envelope protein.

Methods to Produce the Pseudotyped Particles

[0153] This invention also provides methods to prepare a pseudotyped viral particle by transducing a packaging cell line, as described herein with one or both vectors described above, the envelope plasmid and the packaging plasmid under conditions that facilitate packaging of the vector(s) into the envelope particle. In one aspect, the pseudotyped viral particle is a pseudotyped viral particle. In a further aspect, the particles are separated from the cellular supernatant and conjugated to an antibody for cell-specific targeting.

[0154] In one aspect, the complete vector particle is a viral, or alternatively a retroviral vector pseudotyped with a Sindbis virus glycoprotein envelope containing the ZZ domain of Protein A from Staphylococcus aureus or VSVG.

[0155] The genetic information of the viral vector particle is RNA which contains, on the 5' and 3 ' ends, the minimal LTR regions required for integration of the vector. In between the two LTR regions is the psi region which is required for packaging of the vector RNA into the particle. This region is followed by the RRE and cPPT sequences which enhance vector production by transporting the full length vector transcript out of the nucleus for efficient packaging into the vector particle. Next is the polymerase-II promoter MNDU3 which drives the expression of the Factor VIII gene. If necessary, the EGFP gene (enhanced Green Fluorescent Protein) which is driven by the polymerase II PGK promoter is present in the vector. The EGFP gene is used as a reporter gene to detect transduced cells. The above listed genetic elements are transcribed into a full length RNA molecule which is packaged into the vector particle and contains all of the genetic information that will be integrated into the transduced cells.

[0156] The full length RNA transcript is packaged inside the capsid of the vector particle which contains the nucleocapsid, capsid, and matrix proteins which are generated from the packaging plasmid delta-8.91 . The reverse transcriptase polymerse which is generated from the packaging plasmid delta-8.91 is also located within the capsid with the RNA transcript. The capsid encases and protects the full length RNA transcript.

[0157] Surrounding the capsid/RNA complex is the Sindbis-ZZ glycoprotein envelope which is generated from the Sindbis-ZZ plasmid.

[0158] The vector particle can be generated by a transient transfection protocol which includes a packaging cell line (HEK-293T cells), a lipofection reagent (Trans it-293T), and the three plasmids encoding the parts of the vector particle ((pCMV-delta-8.9 (packaging plasmid)), Factor VIII- containing vectors described herein (viral vector plasmid), and pMDG-VSV(envelope plasmid).

[0159] Once the vector particle buds from the packaging cells and is released into the supernatant, this vector particle is conjugated to an antibody as defined herein.

Isolated Host Cells

[0160] Yet further provided is an isolated cell or population of cells, comprising, or alternatively consisting essentially of, or yet further consisting of, a retroviral particle of this invention, which in one aspect, is a viral particle. In one aspect, the isolated host cell is a packaging cell line. In one aspect the cell is a stem cell isolated from chorionic villus tissue from early gestational placenta, e.g., a mesenchymal stem cell (MSC), a hemaptoieitc stem cell (HSC) or an epithelial progenitor cell or an endothelial progenitor cell. When used therapeutically, the cells can be allogeneic or autologous to the subject to be treated. The subjects can be mammalian, e.g., murine, canine, bovine, equine, ovine, feline or a human subject or patient. The isolated cell can be from any appropriate species, e.g., mammalian, e.g., murine, human, canine, bovine, feline, equine, simian, etc.

[0161] In one aspect, the present disclosure provides the C-MSC isolation, expansion and characterization protocol. Applicants have successfully harvested, isolated and propagated C-MSCs from pre-term placenta. To harvest C-MSCs, explant culture was preformed with 20-60 mg of dissected chorionic villus tissue, analogous to the amount of tissue obtained from chorionic villus sampling (CVS). An optimized culture medium was developed comprising, or alternatively consisting of, or yet further consistin of Dulbecco's Modified Eagle Media (DMEM), about 5% prescreened high quality Fetal Bovine Serum (FBS), about 20 ng/ml Fibroblast Growth Factor (FGF) and about 20 ng/ml Epidermal Growth Factor (EGF), that maintains C-MSC stem cell marker expression for at least 15 passages in vitro. Adherent cells grew in spindle-shaped morphology. 1 x 10⁶ cells can be obtained reliably by the third passage, normally occurring before 4 weeks in culture, which demonstrates C-MSCs have greater expansion potential compared to adult MSCs and third trimester placenta chorionic stem cells. Based on Applicants' preliminary analysis of 3 cell lines, C-MSCs express MSC markers CD105, CD90, CD73, CD44, and CD29, and do not express CD 184, HLA-DR as well as hematopoietic and endothelial markers CD45, CD34 and CD31. In addition or alternatively, the cells express the markers CD45, CD34 and/or CD31 and in one aspect, are isolated from early gestation placenta. The C-MSCs are multipotent and can be successfully induced into mesodermal lineages (osteogenic, adipogenic and chondrogenic).

[0162] This invention also provides an population of cells, a population of cells, an expanded population of cells, a substantially homogenous population of cells, a clonal population of cells, as described above.

[0163] This invention further provides an isolated cell or an enriched population of cells that are derived from the stem cell described above. These cells are useful to treat and/or prevent Hemophilia A infection in a subject in need thereof. The cells can be autologous or allogeneic to the subject. The cells can be any mammalian species, e.g., a human, a canine, a feline, a bovine, an equine, an ovine or a murine. In one aspect, the subject is a human patient and is a fetus.

Compositions, Screens and Therapeutic Uses

[0164] Also provided by this invention is a composition or kit comprising any one or more of the compositions described above and a carrier, e.g., an isolated cell, an population of cells, vectors, packaging system, pseudotyped viral, viral particle which in turn may optionally be conjugated to a cell. In one aspect, the carrier is a pharmaceutically acceptable carrier. These compositions can be used diagnostically or therapeutically as described herein and can be used in combination with other known therapies.

[0165] The compositions can be used in vitro to screen for small molecules and other agents that may modify or augment the therapy and replication by adding to the composition varying amounts of the agent to be tested and comparing it to a companion system that does not have the agent but which exhibits the desired therapeutic effect. Alternatively, one can test agents in the viral particle system itself to determine if the agent acts competitively, additively or synergistically with the viral particle system. After an in vitro screen, the test agent or combination therapy can be assayed in an appropriate animal model.

[0166] When the cells, particles and/or antibody conjugated cells (to the particles) are administered to an appropriate animal subject, the animal subject can be used as an animal model to test alternative therapies in the same manner as the in vitro screen. This invention also provides a method to express Factor VIII and/or von Willebrand factor in vivo or ex vivo, comprising, or alternatively consisting essentially of, or yet further consisting of

administering to a subject in need thereof an effective amount of the isolated cells, enriched population of cells, pseudotyped viral particle or the pseudotyped viral particle.

[0167] In another aspect, a method to treat Hemophilia A in a subject such as a mammal is provided, the method comprising, or alternatively consisting essentially of, or yet further consisting of administering to a subject in need thereof an effective amount of the isolated cell or enriched population of cells having inserted therein a polynucleotide encoding function Factor VIII polypeptide. In one aspect, the isolated cell is a pre-term placenta- derived stem cell (also referred to placenta-derived multipotent stem cells). In another aspect, the isolated cell is a pre-term chorionic villus stem cell (C-MSC) comprising exogenously added (transduced) nucleic acid encoding the functional Factor VIII

polypeptide. In a further aspect, the cells are mesenchymal stem cells, hemaptoieitc stem cells (HSCs) or epithelial progenitor cells (endothelial progenitor cells), or a C-MSC express one or more, or two or more, or three of more, or four or more or all five of the markers CD 105, CD90, CD73, CD44, and CD29, and/or does not express one or more of one or more, or two or more, or three or more, or four or more, or all five of CD184, HLA-DR, CD45, CD34 and CD3. In addition or alternatively, the cells express the markers CD45, CD34 and/or CD31 and in one aspect, are isolated from early gestation placenta. The cells are administered in utero.

[0168] In one aspect, the subject is a fetus, such as a human fetus. In another aspect, the isolated C-MSC is autologous to the subject and were isolated from the mother carrying the fetus to be treated. The method can be combined with a pre-term diagnostic to determine if the fetus is likely to develop Hemophilia A prior to the administration of the therapy. Such methods are known in the art. In one aspect, about 1 to up to about 20 million cells per fetus or around about 5 to about 100 million cells per kilogram of estimate fetal weight (the estimate fetal weight is about 0.3kg). The cells can be administered in the range of early gestation of at as early as 5, or 6, or 7 or 8, or 9, or 10, or 1 1 or 12 weeks, or as early as 15 weeks to 25, or alternatively 22 weeks post-conception. In one aspect, the cells are administered at about 16 to 19 weeks post-conception.

[0169] Having been generally described herein, the follow examples are provided to further illustrate this invention.

Examples

Example 1

A. Developing New Treatments for Hemophilia A: Stem Cell-Based Gene Therapy

[0170] As a monogenic disorder with a broad therapeutic window, Hemophilia A is considered as an ideal target for gene or cell-based gene therapy (Roybal, J.L. et al. (2010) Semin Fetal Neonatal Med 15(1 ):46-51 ). A recent landmark clinical trial in hemophilia B demonstrated sustained FIX expression in two-thirds of the patients who underwent in situ gene therapy (Nathwani, A.C. et al. (201 1) N Engl J Med. 365(25):2357-2365). However, in situ gene therapy has not been used for HA because the gene sequence that encodes FVIII is too large for commonly used viral vectors (Miao, H.Z. et al. (2004) Blood 103(9):3412- 3419). Additionally, in situ gene therapy carries potential risks, as gene insertion can cause deleterious mutations, and patients can develop a negative immune response to the viral vector or foreign protein. Ex vivo stem cell based gene therapy avoids direct administration of the viral vector, circumventing these drawbacks.

B. Establishing the Optimal Stem Cell-Based Gene Therapy for Hemophilia A

[0171] Approximately 75% of HA patients are born into families with a history of

Hemophilia A ("HA"). This simplifies prenatal diagnosis, and could allow clinicians to treat fetal patients in utero, preventing the onset of disease and irreversible complications. In previous studies, postnatal xenogeneic transplantation of human bone marrow-derived mesenchymal stem cells (BM-MSCs) has been used to correct the HA phenotype in mice and sheep (Porada, CD. et al. (201 1 ) Exp Hematol. 39(12): 1 124- 1 135; Wang, Q. et al. (2013) J Genet Genomics 40( 12):617-628). However, FVIII expression was transient, possibly due to low engraftment and development of inhibitory antibodies. Prenatal stem cell based gene therapy may provide long term engraftment due to the unique ontogenic opportunities of the fetal environment (Flake, A.W. (2004) Best Pract Res Clin Obstet Gynaecol. 1 8(6):941 -958; Tiblad, E. et al. (2008) Best Pract Res Clin Obstet Gynaecol. 22( 1 ): 189-201). The effect of a small dose of cells will be greater in a fetal patient due to the fetus' small size, and the immunologic naivete of the immature hematopoietic system may enable tolerance induction, preventing inhibitory FVIII antibody formation (Nijagal, A. et al. (201 1 ) J Vis Exp. 47:2303). Therefore, in utero stem cell based-gene therapy holds great promise for HA treatment, since it could induce lifelong tolerance to FVIII (Gaunt, G. et al. (2001) Am J PerinatoL 18(6):299- 312), overcoming the immune-related hurdles currently hindering HA treatment.

[0172] Chorionic villus sampling (CVS) is a prenatal diagnostic technique in which fetal tissue from the placenta is obtained at relatively low risk to the fetus during the first trimester. CVS can be used to obtain autologous stem cells for in vitro manipulation and in utero transplantation prior to the maturation of the fetal immune system (Takahama, Y. (2006) Nat Rev Immunol. 6(2): 127-135). Applicants have established a protocol to isolate and expand early gestation human placental chorionic tissue-derived multi-potent stem cells (CMSCs) (Lankford, L. et al. (2015) World Journal of Stem Cells 7(1): 195-207). The research demonstrated that C-MSCs are analogous to BM-MSCs in terms of plastic adherence, differentiation potential and stem cell marker expression.

[0173] The first trimester of pregnancy represents an ideal semi-allogeneic graft tolerance model, where the fetus is protected from attack by the maternal immune system. During gestation, prior to the maturation of T cells, is a window of opportunity where donor cells may be recognized as self, therefore inducing tolerance to foreign cells (Takahama, Y. (2006) Nat Rev Immunol. 6(2): 127- 135; Tse, D.B. et al. (2005) Fetal Diagn Ther. 20(3): 175- 181 ). This approach will allow prenatal introduction of gene modified stem cells without rejection. As long term engraftment of FVIII secreting cells is required for sustained FVIII expression, eventual human clinical trials will benefit from the use of an autologous cell source administered before the development of the fetal immune system. Sources for autologous stem cells that can be obtained safely during pregnancy are limited. Recent studies showed that MSC-like stem cells exist within the amniotic fluid (AFSC) and they can be obtained through amniocentesis (De Coppi, P. et al. (2007) Nat Biotechnol. 25(1 ): 100-106). However, amniocentesis is usually performed at the second trimester of gestation, which makes AFSCs less suitable to the prenatal therapeutic timeline, as the development of the fetal immune system is already well underway (Roybal, J.L. et al. (2010) Semin Fetal Neonatal Med 15(1):46-51 ; Kunisaki, S.M. et al. (2006) Journal of Pediatric Surgery 41 :675-682;

Golombeck, K. et al. (2006) Am J Obstet Gynecol. 194(3):834-839). [0174] First trimester chorionic villus tissue is source of autologous stem cells for in utero treatment of HA. For pre-human trials, human C-MSCs are transplanted into an immune deficient (NSG) mouse model to test long-term engraftment. C-MSCs cultured from transgenic HA mice are modified to express FVIII, and transplant them into fetal HA mice in utero to test engraftment and phenotype correction. This treatment is designed to cure HA before birth by transplanting modified autologous stem cells, laying the groundwork for the treatment of other monogenic conditions that cause premature death and contribute to childhood morbidity.

C. C-MSC isolation, expansion and characterization

[0175] Applicants have successfully established a protocol to harvest, isolate and propagate C-MSCs from CVS-sized samples of human pre-term placenta (FIGS. 1A-1C). To test the feasibility of obtaining large numbers of C-MSCs from CVS-samples within a clinically relevant timeframe, Applicants perform explant culture with 20-60 mg of dissected early gestational chorionic villus tissue, analogous to the amount of tissue obtained from CVS. Applicants have developed an optimized culture medium— Dulbecco's Modified Eagle Media (DMEM), 5% prescreened high quality Fetal Bovine Serum (FBS), 20 ng/ml Fibroblast Growth Factor (FGF) and 20 ng/ml Epidermal Growth Factor (EGF). Adherent cells grew in spindle-shaped morphology. 10⁶ cells can be obtained reliably by the third passage, normally occurring before 4 weeks in culture. Previous studies have shown that C- MSCs have greater expansion potential compared to adult MSCs and third trimester placenta chorionic stem cells (Guillot, P.V. et al. (2007) Stem Cells 25(3):646-654; Jones, G.N. et al. (2012) PLoS One 7(9):e43395). Based on this analysis of 3 cell lines, C-MSCs express MSC markers CD105, CD90, CD73, CD44, and CD29, and do not express CD184, HLA-DR as well as hematopoietic and endothelial markers CD45, CD34 and CD31. Applicants' data shows that C-MSCs are multipotent and can be successfully induced into mesodermal lineages (osteogenic, adipogenic and chondrogenic) (FIGS. 2A-2B).

D. C-MSCs can be efficiently transduced by a lentiviral vector containing FVIII

[0176] C-MSCs were transduced with a lentiviral vector encoded with B-domain deleted human FVIII (hFVIII), GFP and luciferase driven by MNDU3promoter (pCCLc-MNDU3- EGFP/LUC-PGK-Fb-WPRE). The efficiency of transduction was determined by gpf expression to be 80-90% (FIGS. 3A-3B). To confirm that GFP+ C-MSCs produce hFVIII, Applicants performed immunocytochemical staining for hFVIII (FIG. 4). C-MSCs were positive for hFVIII indicating that gene-modified C-MSCs expressed FVIII in vitro.

E. Transduced C-MSCs secrete hFVIII in cell supernatant

[0177] To evaluate whether hFVIII expression by transduced C-MSCs directly correlates with hFVIII secretion, Applicants used an immunocapture hFVIII enzyme-linked

immunosorbent assay (ELISA) to quantify active FVIII levels in the supernatant. ELISA was performed on supernatants of transduced four C-MSC lines according to the manufacturer's instructions. Briefly, hFVIII concentration was measured before transduction, 72 hours, two weeks, four weeks, and six weeks post transduction. Following transduction, the

concentration of hFVIII in the supernatant was approximately 24 ng/10⁶ cells with slight variations at each time point. Phosphate Buffer Saline (PBS) and supernatants of pre- transduction C-MSCs were used as negative controls; there was no detectable hFVIII secretion. This data indicates efficient transduction of C-MSCs and FVIII transgene expression of the FVIII antigen in the cell supernatant.

F. Therapeutic levels of FVIII coagulation activity in FVIII-transduced C-MSCs

[0178] Applicants analyzed the biologically active FVIII coagulation activity (FVIILC) by performing a FVIII: C chromogenic assay on cell supernatants from transduced C-MSCs. The standard plasma FVIILC used as control was approximately 200%. In the supernatant of transduced C-MSCs, hFVIII activity was approximately 4.5IU/10⁶ (~150% FVIII activity/10⁶). The hFVIII activity level in cell supernatant of pre-transduction C-MSCs or PBS had no detectable hFVIII activity. This data indicates therapeutic levels of FVIILC in the cell supernatant.

G. Transduced C-MSCs can be detected in vivo after cell transplantation

[0179] C-MSCs, l l O⁶ were transduced with GFP-luciferase and transplanted in utero via intraperitoneal route of first trimester embryonic age 12.5 day fetus using a 33 gauge non- coring needle. Bioluminescence imaging analysis performed in the fetuses and 5 days postnatal pup revealed focal density and transgene expression one of pups (FIGS. 5A-5B) clearly indicating that we can effectively, safely transplant and track gene-modified C-MSCs in utero via intraperitoneal route.

[0180] To summarize, ths presented data show that C-MSCs are expandable, express MSC markers, are multipotent, and can be efficiently transduced to express functional FVIII. In vivo detection of luciferase expressing gene-modified C-MSCs demonstrate that the disclosed in utero transplantation protocol is effective.

Example 2

A. Establishing the viral vectors for efficient transduction in C-MSCs

[0181] Applicant designed the following additional vectors for C-MSC transduction to improve the transduction efficiency and consistency:

Vector 1. FVIII-NEO: pCCLc-MNDU3-F8-PGK-NEO-WPRE.

Vector 2. X-NEO: pCCLc-MNDU3-X-PGK-NEO-WPRE.

Vector 3. LUC/GFP: pCCLc-M DU3-LUC-PGK-EGFP-WPRE

[0182] All vectors were synthesized and tittered by the Vector Core at the University of California Davis Medical Center (UCDMC Stem Cell Program). A killing assay for antibiotic selection using various doses of G418 was performed on C-MSCs and 200 μg/ml was found to be the optimal dosage to use that killed all the non-transduced cells after 7 days of incubation.

B. Optimizing the viral transduction in C-MSCs

[0183] To determine the optimal multiplicity of infection (MOI) to use for maximum FVffl secretion, C-MSCs were transduced with either X-NEO or FVIII-NEO at different MOI and incubated for 72h. FVIII activity in the media was assessed using chromogenic assay. MOI of 10 was found to be optimal with an activity reaching 1.1 IU/106 cells and was used for all further experiments. (FIG. 6). FVIII expression in all the 4 cell lines transduced with either X-NEO vector (negative control) or FVIII-NEO vector was confirmed by RT-PCR (FIG. 7) and Western blot (FIG. 8) and activity by chromogenic assay (FIG. 9). All the four cell lines expressed high levels of active Factor VIII when compared to control.

Equivalents

[0184] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

[0185] The present technology illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising," "including," "containing," etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the present technology claimed.

[0186] Thus, it should be understood that the materials, methods, and examples provided here are representative of preferred aspects, are exemplary, and are not intended as limitations on the scope of the present technology.

[0187] The present technology has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the present technology. This includes the generic description of the present technology with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0188] In addition, where features or aspects of the present technology are described in terms of Markush groups, those skilled in the art will recognize that the present technology is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0189] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, to the same extent as if each were incorporated by reference individually. In case of conflict, the present specification, including definitions, will control.

[0190] Other aspects are set forth within the following claims.

References

1. Bolton-Maggs, P.H. and K.J. Pasi, Haemophilias A and B. Lancet, 2003.

361(9371): 1801-9.

2. Kulkarni, R. et al., Sites of initial bleeding episodes, mode of delivery and age of diagnosis in babies with haemophilia diagnosed before the age of 2 years: a report from The Centers for Disease Control and Prevention 's (CDC) Universal Data Collection (UDC) project. Haemophilia, 2009. 15(6): 1281 -90.

3. Manco- Johnson, M.J. et al., Prophylaxis versus episodic treatment to prevent joint disease in boys with severe hemophilia. N Engl J Med, 2007. 357(6):535-44.

4. Chalmers, E.A. et al., Early factor VIII exposure and subsequent inhibitor development in children with severe haemophilia A. Haemophilia, 2007. 13(2): 149-55.

5. Roybal, J.L., M.T. Santore, and A.W. Flake, Stem cell and genetic therapies for the fetus. Semin Fetal Neonatal Med, 2010. 15(1 ):46-51 .

6. Nathwani, A.C. et al., Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med, 201 1. 365(25):2357-65.

7. Miao, H.Z. et al., Bioengineering of coagulation factor VIII for improved secretion. Blood, 2004. 103(9):3412-9.

8. Porada, CD. et al., Phenotypic correction of hemophilia A in sheep by postnatal intraperitoneal transplantation of FVIII-expressing MSC. Exp Hematol, 201 1. 39(12): 1 124- 1 135 e4.

9. Wang, Q. et al., The mesenchymal stem cells derived from transgenic mice carrying human coagulation factor VIII can correct phenotype in hemophilia A mice. J Genet Genomics, 2013. 40(12):617-28.

10. Flake, A. W., In utero stem cell transplantation. Best Pract Res Clin Obstet Gynaecol, 2004. 18(6):941-58.

1 1. Tiblad, E. and M. Westgren, Fetal stem-cell transplantation. Best Pract Res Clin Obstet Gynaecol, 2008. 22(1 ): 189-201.

12. Nijagal, A. et al., A mouse model of in utero transplantation. J Vis Exp, 201 1(47).

13. Gaunt, G. and K. Ramin, Immunological tolerance of the human fetus. Am J

Perinatol, 2001. 18(6):299-312. 14. Takahama, Y., Journey through the thymus: stromal guides for T-cell development and selection. Nat Rev Immunol, 2006. 6(2): 127-35.

15. L. Lankford, S.T., Becker, J., Ryzhuk, V., Long, C, Farmer, D.L., Wang, A, Early gestation chorionic villi-derived stromal cells for fetal tissue engineering. World Journal of Stem Cells (Accepted for publication), 2014.

16. Tse, D.B. et al., Heterogeneity in fetal immunocompetence during the second trimester of gestation. Implications for treatment of nonimmune genetic disorders by in utero transplantation. Fetal Diagn Ther, 2005. 20(3): 175-81.

17. De Coppi, P. et al., Isolation of amniotic stem cell lines with potential for therapy. Nat Biotechnol, 2007. 25(l): 100-6.

18. Shaun M. Kunisaki, D.A.F., Dario O. Fauza, Fetal tracheal reconstruction with cartilaginous grafts engineered from mesenchymal amniocytes. Journal of Pediatric Surgery, 2006. 41:675-682.

19. Golombeck, K. et al., Maternal morbidity after maternal-fetal surgery. Am J Obstet Gynecol, 2006. 194(3):834-9.

20. Guillot, P.V. et al., Human first-trimester fetal MSC express pluripotency markers and grow faster and have longer telomeres than adult MSC. Stem Cells, 2007. 25(3):646-54.

21. Jones, G.N. et al., Ontological differences in first compared to third trimester human fetal placental chorionic stem cells. PLoS One, 2012. 7(9):e43395.

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23. Guillot, P.V. et al., Intrauterine transplantation of human fetal mesenchymal stem cells from first-trimester blood repairs bone and reduces fractures in osteogenesis imperfecta mice. Blood, 2008. 111(3): 1717-25.

24. Westgren, M. et al., Lack of evidence of permanent engraftment after in utero fetal stem cell transplantation in congenital hemoglobinopathies. Transplantation, 1996. 61(8): 1 176-9.

25. Rossant, J. and J.C. Cross, Placental development: lessons from mouse mutants. Nat Rev Genet, 2001. 2(7):538-48.

Claims

WHAT IS CLAIMED IS:

1. A method to treat a genetic disorder or disease characterized by lack of expression of a functional protein or polypeptide in a subject in need thereof, comprising administering to the subject an effective amount of an isolated cell expressing the functional protein or polypeptide, thereby treating the genetic disorder or disease.

2. The method of claim 1, wherein the subject is a fetus.

3. The method of claim 2, wherein the fetus is a human fetus.

4. The method of claim 1, wherein the genetic disease or disorder is selected from the group of Hemophilia A, Duchenne's Muscular Dystrophy, osteogenesis imperfecta (01), thalassemia, sickle cell anemia, cystic fibrosis, spinal muscular atrophy, severe intrauterine growth restriction, or MPS.

5. The method of claim 1, further comprising determining if the fetus is likely to develop the genetic disorder or disease prior to the treatment.

6. A vector comprising:

(a) a backbone comprising essential sequences for integration into a target cell genome;

(b) a nucleic acid encoding a functional polypeptide; and

(c) a first expression control element that regulates expression of the nucleic acid encoding the functional polypeptide.

7. The vector of claim 6, wherein the fuctional polypeptide is Factor VIII polypeptide for the treatment of Hemophilia A.

8. The vector of claim 7 further comprising a polynucleotide encoding functional von Willebrand factor and an expression control element that regulates expression of the polynucleotide encoding functional von Willebrand factor.

9. The vector of claim 7, wherein the first expression control element is a Polymerase II promoter, optionally MNDU3 or a phosphoglycerate kinase (PGK) promoter.

10. The vector of claim 6 or 7, further comprising a nucleic acid encoding a detectable marker.

11. The vector of claim 10, wherein the detectable marker comprises green fluorescent protein (GFP) or luciferase (LUC) optionally under the control of second expression control element.

12. The vector of claim 11, wherein the second expression control element comprises a Polymerase II promoter.

13. The vector of claim 12, wherein the Polymerase II promoter comprises a

phosphoglycerate kinase (PGK) promoter.

14. The vector of any of claim 6 or 7, further comprising an enhancer element.

15. The vector of claim 14, wherein enhancer element comprises WPRE.

16. The vector of claim 7, wherein the vector comprises the vector contained within SEQ ID NO: 1, 2 or 5, or an equivalent thereof, or a polynucleotide having at least 80 % identity thereto.

17. A viral packaging system comprising:

(a) the vector of claim 6 or 7, wherein the backbone is derived from a virus;

(b) a packaging plasmid; and

(c) an envelope plasmid.

18. The viral packaging system of claim 17, wherein the envelope plasmid is selected from the group of: a plasmid comprising a S. aureus ZZ domain sequence,a sequence encoding a pseudotyped envelope protein or a sequence encoding a VSVG envelope, a VSV sequence, or a pMD.G VSV sequence.

19. The viral packaging system of claim 18, further comprising (d) a packaging cell line.

20. The viral packaging system of claim 19, wherein the packaging cell line is HEK-293 cell.

21. A method for producing a pseudotyped viral particle, comprising transducing a packaging cell line with the system of claim 17 under conditions suitable to package the viral vector.

22. The method of claim 21, wherein the packaging cell line is HEK-293.

23. A pseudotyped viral particle produced by the method of claim 22.

24. A pseudotyped viral particle comprising a viral vector comprising a polynucleotide encoding a functional polypeptide and an envelope protein comprising a polypeptide selected from the group of ZZ S. aureus domain or a VSV domain.

25. The viral particle of claim 24, wherein the functional polypeptide is Factor VIII.

26. The pseudotyped viral of claim 24 or 25 further conjugated to an isolated cell.

27. An isolated cell comprising the vector of claim 6 or 7, or the pseudotyped viral particle of claim 24 or 25.

28. The isolated cell of claim 27, wherein the isolated cell is a stem cell, a mesenchymal stem cell, a hemaptoieitc stem cells (HSC), an epithelial progenitor cell or an edothelial progenitor cell isolated from placenta.

29. The isolated cell of claim 28, wherein the cell is a mesenchumal stem cell and expresses a marker profile of the group: expresses one or more of the markers CD105, CD90, CD73, CD44, and CD29, and does not express one or more of CD184, HLA-DR, CD45, CD31, or CD34; or expresses one or more of the markers CD105, CD90, CD73, CD44, and CD29 and does not express one or more CD45, CD31, or CD34; or does express one or more of CD45, CD31 and CD34.

30. A population of cells claim 24 or 25.

31. A substantially homogenous population of claim 30.

32. A clonal population of cells of claim 31.

33. An expanded population of cell derived from the isolated cell of claim 30.

34. The expanded population of claim 33, wherein the derived population of cells comprises a population of the mesodermal lineage.

35. The expanded population of claim 34, wherein the cell of mesodermal lineage comprises an osteogenic lineage, an adipogenic lineage, or an chondrogenic lineage.

36. A composition comprising a cell of claim 27, and a carrier.

37. The composition of claim 36, wherein the carrier is a pharmaceutically acceptable carrier.

38. A method to express a functional polypeptide comprising culturing the cell of claim 27 under conditions that favor expression of the polypeptide.

39. The method of claim 38, further comprising isolating the polypeptide prepared by the method of claim 38.

40. The method of claim 38 or 39, wherein the isolated cell is a mammalian cell, a stem cell or an iPSC cell.

41. A method to treat or inhibit Hemophilia A in a subject in need thereof, comprising administering to the subject in need thereof an effective amount of the isolated cell expressing Factor VII, thereby treating Hemophilia A in the subject.

42. The method of claim 41, wherein the subject is a fetus.

43. The method of claim 42, wherein the fetus is a human fetus.

44. The method of claim 41, further comprising determining if the fetus is likely to develop Hemophilia A prior to the administration of the therapy.

45. Use of the cell expessing functional Factor VIII in the manufacture of a medicament to treat Hemophilia A in a subject in need thereof.

46. The use of claim 45, wherein the cell is a human cell, a human stem cell, a human iPSC cell, that is autologous or allogenic to the subject.

47. The use of claim 45 or 46, wherein the subject is a fetus.