Biopolym. Cell. 2013; 29(1):21-32.
Stem cell – based gene therapy
1Nazir Z., 1Irshad S.
  1. Institute of Biochemistry and Biotechnology, University of the Punjab
    Lahore, Pakistan


Stem cells have huge potential for regenerative medicine. Adult stem cell (HSC)-based therapies have been proved to be safe and efficient for several decades, and adult MSC therapies are showing efficacy in some experiments while in other trials mixed results are obtained such as only short lived effects due to poor cellular retention or other reasons that have to be further tested. Although iPSCs might suggest a great hope for the stem cell therapy, still there are important safety issues to be considered before these cells are marketed for clinical trials. However, the advanced potential to generate stem cell lines, matched to a particular patient, and to perform homologous gene correction or targeted transgene insertion into a safe dock site in the genome prior to further expansion and differentiation offer great prospects for future regenerative medicine. Furthermore, the development of the recombinant adeno-associated virus (rAAV) technology and the use of the Zinc Finger Nuclease (ZFN) technology are promoting the homologous recombination as a best possible tool for stem cell-based gene therapy.
Keywords: Adult stem cells, MSCs, iPSCs, ZFN, rAAV, homologous gene correction, regenerative medicine


[1] Mullen C. A., Snitzer K., Culver K. W., Morgan R. A., Anderson W. F., Blaese R. M. Molecular analysis of T lymphocyte-directed gene therapy for adenosine deaminase deficiency: long-term expression in vivo of genes introduced with a retroviral vector Hum. Gene. Ther 1996 7, N 9:1123–1129.
[2] Hacein-Bey-Abina S., Le Deist F., Carlier F., Bouneaud C., Hue C., De Villartay J. P., Thrasher A. J., Wulffraat N., Sorensen R., Dupuis-Girod S., Fischer A., Davies E. G., Kuis W., Leiva L., Cavazzana-Calvo M. Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene therapy N. Engl. J. Med 2002 346, N 16:1185–1193.
[3] Hacein-Bey-Abina S., von Kalle C., Schmidt M., Le Deist F., Wulffraat N., McIntyre E., Radford I., Villeval J. L., Fraser C. C., Cavazzana-Calvo M., Fischer A. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency N. Engl. J. Med 2003 348, N 3:255–256.
[4] Aiuti A., Slavin S., Aker M., Ficara F., Deola S., Mortellaro A., Morecki S., Andolfi G., Tabucchi A., Carlucci F., Marinello E., Cattaneo F., Vai S., Servida P., Miniero R., Roncarolo M. G., Bordignon C. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning Science 2002 296, N 5577:2410–2413.
[5] Gregory C. A., Prockop D. J., Spees J. L. Non-hematopoietic bone marrow stem cells: molecular control of expansion and differentiation Exp. Cell Res 2005 306, N 2:330–335.
[6] Jiang Y., Jahagirdar B. N., Reinhardt R. L., Schwartz R. E., Keene C. D., Ortiz-Gonzalez X. R., Reyes M., Lenvik T., Lund T., Blackstad M., Du J., Aldrich S., Lisberg A., Low W. C., Largaespada D. A., Verfaillie C. M. Pluripotency of mesenchymal stem cells derived from adult marrow Nature 2002 418, N 6893 P. 41–49.
[7] Stocum D. L. Stem cells in CNS and cardiac regeneration Adv. Biochem. Eng. Biotechnol 2005 93, N 1:135–159.
[8] Brenner M. K., Rill D. R., Holladay M. S., Heslop H. E., Moen R. C., Buschle M., Krance R. A., Santana V. M., Anderson W. F., Ihle J. N. Gene marking to determine whether autologous marrow infusion restores long-term haemopoiesis in cancer patients Lancet 1993 342, N 8880:1134–1137.
[9] Reya T., Duncan A. W., Ailles L., Domen J., Scherer D. C., Willert K., Hintz L., Nusse R., Weissman I. L. A role for Wnt signalling in self-renewal of haematopoietic stem cells Nature 2003 423, N 6938:409–414.
[10] Willert K., Brown J. D., Danenberg E., Duncan A. W., Weissman I. L., Reya T., Yates J. R. 3rd, Nusse R. Wnt proteins are lipid-modified and can act as stem cell growth factors Nature 2003 423, N 6938:448–452.
[11] Evans M. J., Kaufman M. H. Establishment in culture of pluripotential cells from mouse embryos Nature 1981 292, N 5819 P. 154–156.
[12] Martin G. R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells Proc. Natl Acad. Sci. USA 1981 78, N 12:7634– 7638.
[13] Thomson J. A., Itskovitz-Eldor J., Shapiro S. S., Waknitz M. A., Swiergiel J. J., Marshall V. S., Jones J. M. Embryonic stem cell lines derived from human blastocysts Science 1998 282, N 5391:1145–1147.
[14] Russell D. W., Hirata R. K. Human gene targeting by viral vectors Nat. Genet 1998 18, N 4:325–330.
[15] Manno C. S., Pierce G. F., Arruda V. R., Glader B., Ragni M., Rasko J. J., Ozelo M. C., Hoots K., Blatt P., Konkle B., Dake M., Kaye R., Razavi M., Zajko A., Zehnder J., Rustagi P. K., Nakai H., Chew A., Leonard D., Wright J. F., Lessard R. R., Sommer J. M., Tigges M., Sabatino D., Luk A., Jiang H., Mingozzi F., Couto L., Ertl H. C., High K. A., Kay M. A. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response Nat. Med 2006 12, N 3 P. 342–347.
[16] Manno C. S., Chew A. J., Hutchison S., Larson P. J., Herzog R. W., Arruda V. R., Tai S. J., Ragni M. V., Thompson A., Ozelo M., Couto L. B., Leonard D. G., Johnson F. A., McClelland A., Scallan C., Skarsgard E., Flake A. W., Kay M. A., High K. A., Glader B. AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B Blood 2003 101, N 8 P. 2963–2972.
[17] Kohli M., Rago C., Lengauer C., Kinzler K. W., Vogelstein B. Facile methods for generating human somatic cell gene knockouts using recombinant adenoassociated viruses Nucleic Acids Res 2004 32, N 1 e3.
[18] Brenneman M., Gimble F. S., Wilson J. H. Stimulation of intrachromosomal homologous recombination in human cells by electroporation with site-specific endonucleases Proc. Natl Acad. Sci. USA 1996 93, N 8:3608–3612.
[19] Jasin M. Genetic manipulation of genomes with rare-cutting endonucleases Trends Genet 1996 12, N 6:224–228.
[20] Rouet P., Smih F., Jasin M. Introduction of double-strand breaks into the genome of mouse cells by expression of rarecutting endonuclease Mol. Cell. Biol 1994 14, N 12 P. 8096–8106.
[21] Porteus M. H., Carroll D. Gene targeting using zinc finger nucleases Nat. Biotechnol 2005 23, N 8:967–973.
[22] Durai S., Mani M., Kandavelou K., Wu J., Porteus M. H., Chandrasegaran S. Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells Nucleic Acids Res 2005 33, N 18:5978–5990.
[23] Kim Y. G., Cha J., Chandrasegaran S. Hybrid restriction enzymes: zinc finger fusions to Fok I cleavage domain Proc. Natl Acad. Sci. USA 1996 93, N 3:1156–1160.
[24] Kim Y. G., Chandrasegaran S. Chimeric restriction endonuclease Proc. Natl Acad. Sci. USA 1994 91, N 3:883–887.
[25] Kim Y. G., Smith J., Durgesha M., Chandrasegaran S. Chimeric restriction enzyme: Gal4 fusion to FokI cleavage domain Biol. Chem 1998 379, N 4:489–495.
[26] Diakun G. P., Fairall L., Klug A. EXAFS study of the zinc-binding sites in the protein transcription factor IIIA Nature 1986 324, N 6098:698–699.
[27] Pavletich N. P., Pabo C. O. Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A Science 1991 252, N 5007:809–817.
[28] Pabo C. O., Peisach E., Grant R. A. Design and selection of novel Cys2His2 zinc finger proteins Annu. Rev. Biochem 2001 70:313–340.
[29] Segal D. J., Barbas C. F. 3rd. Custom DNA-binding proteins come of age: polydactyl zinc-finger proteins Curr. Opin. Biotechnol 2001 12, N 6:632–637.
[30] Smith J., Bibikova M., Whitby F. G., Reddy A. R., Chandrasegaran S., Carroll D. Requirements for double-strand cleavage by chimeric restriction enzymes with zinc finger DNA-recognition domains Nucleic Acids Res 2000 28, N 17:3361–3369.
[31] Bibikova M., Carroll D., Segal D. J., Trautman J. K., Smith J., Kim Y. G., Chandrasegaran S. Stimulation of homologous recombination through targeted cleavage by chimeric nucleases Mol. Cell. Biol 2001 21, N 1:289–297.
[32] Porteus M. H., Cathomen T., Weitzman M. D., Baltimore D. Efficient gene targeting mediated by adeno-associated virus and DNA double-strand breaks Mol. Cell. Biol 2003 23, N 10 P. 3558–3565.
[33] Porteus M. H. Mammalian gene targeting with designed zinc finger nucleases Mol. Ther 2006 13, N 2:438–446.
[34] Urnov F. D., Miller J. C., Lee Y. L., Beausejour C. M., Rock J. M., Augustus S., Jamieson A. C., Porteus M. H., Gregory P. D., Holmes M. C. Highly efficient endogenous human gene correction using designed zinc-finger nucleases Nature 2005 435, N 7042:646–651.
[35] Miller D. G., Petek L. M., Russell D. W. Human gene targeting by adenoassociated virus vectors is enhanced by DNA doublestrand breaks Mol. Cell. Biol 2003 23, N 10:3550– 3557.
[36] Parekkadan B., Milwid J. M. Mesenchymal stem cells as therapeutics Annu. Rev. Biomed. Eng 2010 12:87–117.
[37] Thomas E. D., Ashley C. A., Lochte H. L. Jr., Jaretzki A. 3rd, Sahler O. D., Ferrebee J. W. Homografts of bone marrow in dogs after lethal total-body radiation Blood 1959 14, N 6 P. 720–736.
[38] Copelan E. A. Hematopoietic stem-cell transplantation N. Engl. J. Med 2006 354, N 17:1813–1826.
[39] Friedenstein A. J., Chailakhyan R. K., Latsinik N. V., Panasyuk A. F., Keiliss-Borok I. V. Stromal cells responsible for transferring the microenvironment of the hemopoietic tissues. Cloning in vitro and retransplantation in vivo Transplantation 1974 17, N 4:331–340.
[40] Ankrum J., Karp J. M. Mesenchymal stem cell therapy: Two steps forward, one step back Trends. Mol. Med 2010 16, N 5 P. 203–209.
[41] Karp J. M., Leng Teo G. S. Mesenchymal stem cell homing: The devil is in the details Cell. Stem. cell 2009 4, N 3:206–216.
[42] Meyerrose T., Olson S., Pontow S., Kalomoiris S., Jung Y., Annett G., Bauer G., Nolta J. A. Mesenchymal stem cells for the sustained in vivo delivery of bioactive factors Adv. Drug. Deliv. Rev 2010 62, N 12:1167–1174.
[43] Capoccia B. J., Robson D. L., Levac K. D., Maxwell D. J., Hohm S. A., Neelamkavil M. J., Bell G. I., Xenocostas A., Link D. C., Piwnica-Worms. D., Nolta J. A., Hess D. A. Revascularization of ischemic limbs after transplantation of human bone marrow cells with high aldehyde dehydrogenase activity Blood 2009 113, N 21:5340–5351.
[44] Le Blanc K., Frassoni F., Ball L., Locatelli F., Roelofs H., Lewis I., Lanino E., Sundberg B., Bernardo M. E., Remberger M., Dini G., Egeler R. M., Bacigalupo A., Fibbe W., Ringden O. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study Lancet 2008 371, N 9624:1579–1586.
[45] Aggarwal S., Pittenger M. F. Human mesenchymal stem cells modulate allogeneic immune cell responses Blood 2005 105, N 4:1815–1822.
[46] Bauer G., Dao M. A., Case S. S., Meyerrose T., Wirthlin L., Zhou P., Wang X., Herrbrich P., Arevalo J., Csik S., Skelton D. C., Walker J., Pepper K., Kohn D. B., Nolta J. A. In vivo biosafety model to assess the risk of adverse events from retroviral and lentiviral vectors Mol. Ther 2008 16, N 7:1308–1315.
[47] Ripa R. S., Haack-Sorensen M., Wang Y., Jorgensen E., Mortensen S., Bindslev L., Friis T., Kastrup J. Bone marrow derived mesenchymal cell mobilization by granulocyte-colony stimulating factor after acute myocardial infarction: results from the stem cells in myocardial infarction (STEMMI) trial Circulation 2007 116, N 11 (suppl.) I–24–I–30.
[48] Joyce N., Annett G., Wirthlin L., Olson S., Bauer G., Nolta J. A. Mesenchymal stem cells for the treatment of neurodegenerative disease Regen. Med 2010 5, N 6:933–946.
[49] Brouard N., Chapel A., Thierry D., Charbord P., Peault B. Transplantation of gene-modified human bone marrow stromal cells into mouse-human bone chimeras J. Hematother. Stem Cell. Res 2000 9, N 2:175–181.
[50] Ding L., Lu S., Batchu R. B., Saylors R. L. III, Munshi N. C. Bone marrow stromal cells as a vehicle for gene transfer Gene. Ther 1999 6, N 9:1611–1616.
[51] Cavazzana-Calvo M., Payen E., Negre O., Wang G., Hehir K., Fusil F., Down J., Denaro M., Brady T., Westerman K., Cavallesco R., Gillet-Legrand B., Caccavelli L., Sgarra R., MaoucheChretien L., Bernaudin F., Girot R., Dorazio R., Mulder G. J., Polack A., Bank A., Soulier J., Larghero J., Kabbara N., Dalle B., Gourmel B., Socie G., Chretien S., Cartier N., Aubourg P., Fischer A., Cornetta K., Galacteros F., Beuzard Y., Gluckman E., Bushman F., Hacein-Bey-Abina S., Leboulch P. Transfusion independence and HMGA2 activation after gene therapy of human b-thalassaemia Nature 2010 467, N 7313:318–322.
[52] Roobrouck V. D., Ulloa-Montoya F., Verfaillie C. M. Self-renewal and differentiation capacity of young and aged stem cells Exp. Cell Res 2008 314, N 9:1937–1944.
[53] Takahashi K., Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors Cell 2006 126, N 4:663–676.
[54] Wernig M., Zhao J. P., Pruszak J., Hedlund E., Fu D., Soldner F., Broccoli V., Constantine-Paton M., Isacson O., Jaenisch R. Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinson's disease Proc. Natl Acad. Sci. USA 2008 105, N 15:5856–5861.
[55] Hanna J., Wernig M., Markoulaki S., Sun C. W., Meissner A., Cassady J. P., Beard C., Brambrink T., Wu L. C., Townes T. M., Jaenisch R. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin Science 2007 318, N 5858:1920–1923.
[56] Ebert A. D., Yu J., Rose F. F., Mattis V. B., Lorson C. L., Thomson J. A., Svendsen C. N. Induced pluripotent stem cells from a spinal muscular atrophy patient Nature 2009 457, N 7227 P. 277–280.
[57] Park I. H., Arora N., Huo H., Maherali N., Ahfeldt T., Shimamura A., Lensch M. W., Cowan C., Hochedlinger K., Daley G. Q. Disease-specific induced pluripotent stem cells Cell 2008 134, N 5:877–886.
[58] Kambal A., Mitchell G., Cary W., Gruenloh W., Jung Y., Kalomoiris S., Nacey C., McGee J., Lindsey M., Fury B., Bauer G., Nolta J. A., Anderson J. S. Generation of HIV-1 resistant and functional macrophages from hematopoietic stem cell-derived induced pluripotent stem cells Mol. Ther 2011 19, N 3 P. 584–593.
[59] Okita K., Ichisaka T., Yamanaka S. Generation of germline-competent induced pluripotent stem cells Nature 2007 448, N 7151:313–317.
[60] Hacein-Bey-Abina. S., Von Kalle C., Schmidt M., McCormack M. P., Wulffraat N., Leboulch P., Lim A., Osborne C. S., Pawliuk R., Morillon E., Sorensen R., Forster A., Fraser P., Cohen J. I., de Saint Basile G., Alexander I., Wintergerst U., Frebourg T., Aurias A., Stoppa-Lyonnet D., Romana S., Radford-Weiss I., Gross F., Valensi F., Delabesse E., Macintyre E., Sigaux F., Soulier J., Leiva L. E., Wissler M., Prinz C., Rabbitts T. H., Le Deist F., Fischer A., Cavazzana-Calvo M. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1 Science 2003 302, N 5644:415–419.
[61] Gore A., Li Z., Fung H. L., Young J. E., Agarwal S., Antosiewicz-Bourget J., Canto I., Giorgetti A., Israel M. A., Kiskinis E., Lee J. H., Loh Y. H., Manos P. D., Montserrat N., Panopoulos A. D., Ruiz S., Wilbert M. L., Yu J., Kirkness E. F., Izpisua Belmonte J. C., Rossi D. J., Thomson J. A., Eggan K., Daley G. Q., Goldstein L. S., Zhang K. Somatic coding mutations in human induced pluripotent stem cells Nature 2011 471, N 7336:63–67.
[62] Katsara O., Mahaira L. G., Iliopoulou E. G., Moustaki A., Antsaklis A., Loutradis D., Stefanidis K., Baxevanis C. N., Papamichail M., Perez S. A. Effects of donor age, gender, and in vitro cellular aging on the phenotypic, functional, and molecular characteristics of mouse bone marrow-derived mesenchymal stem cells Stem Cells Dev 2011 20, N 9:1549–1561.
[63] Hematti P. Human embryonic stem cell-derived mesenchymal progenitors: an overview Methods Mol. Biol 2011 690 P. 163–174.
[64] Gruenloh W., Kambal A., Sondergaard C., McGee J., Nacey C., Kalomoiris S., Pepper K., Olson S., Fierro F., Nolta J. A. Characterization and in vivo testing of mesenchymal stem cells derived from human embryonic stem cells Tissue. Eng. Part. A 2011 17, N 11–12:1517–1525.
[65] Lian Q., Zhang Y., Zhang J., Zhang H. K., Wu X., Zhang Y., Lam F. F., Kang S., Xia J. C., Lai W. H., Au K. W., Chow Y. Y., Siu C.W., Lee C. N., Tse H. F. Functional mesenchymal stem cells derived from human induced pluripotent stem cells attenuate limb ischemia in mice Circulation 2010 121, N 9:1113– 1123.
[66] Fierro F., Kalomoiris S., Sondergaard C., Nolta J. A. Effects on proliferation and differentiation of multipotent bone marrow stromal cells engineered to express growth factors for combined cell and gene therapy Stem Cells 2011 29, N 11:1727– 1737.
[67] Meyerrose T. E., Roberts M., Ohlemiller K. K., Vogler C. A., Wirthlin L., Nolta J. A., Sands M. S. Lentiviral-transduced human mesenchymal stem cells persistently express therapeutic levels of enzyme in a xenotransplantation model of human disease Stem Cells 2008 26, N 7:1713–1722.
[68] Halme D. G., Kessler D. A. FDA regulation of stem-cell-based therapies N. Engl. J. Med 2006 355, N 16:1730–1735.
[69] Nakagawa M., Koyanagi M., Tanabe K., Takahashi K., Ichisaka T., Aoi T., Okita K., Mochiduki Y., Takizawa N., Yamanaka S. Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts Nat. Biotechnol 2008 26, N 1 P. 101–106.
[70] Hacein-Bey-Abina S., Garrigue A., Wang G. P., Soulier J., Lim A., Morillon E., Clappier E., Caccavelli L., Delabesse E., Beldjord K., Asnafi V., MacIntyre E., Dal Cortivo L., Radford I., Brousse N., Sigaux F., Moshous D., Hauer J., Borkhardt A., Belohradsky B. H., Wintergerst U., Velez M. C., Leiva L., Sorensen R., Wulffraat N., Blanche S., Bushman F. D., Fischer A., Cavazzana-Calvo M. Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1 J. Clin. Invest 2008 118, N 9:3132–3142.
[71] Montini E., Cesana D., Schmidt M., Sanvito F., Ponzoni M., Bartholomae C., Sergi Sergi L., Benedicenti F., Ambrosi A., Di Serio C., Doglioni C., von Kalle C., Naldini L. Hematopoietic stem cell gene transfer in a tumor-prone mouse model uncovers low genotoxicity of lentiviral vector integration Nat. Biotechnol 2006 24, N 6:687–696.
[72] Gonzalez F., Boue S., Izpisua Belmonte J. C. Methods for making induced pluripotent stem cells: reprogramming a la carte Nat. Rev. Genet 2011 12, N 4:231–242.
[73] Zhu S., Li W., Zhou H., Wei W., Ambasudhan R., Lin T., Kim J., Zhang K., Ding S. Reprogramming of human primary somatic cells by OCT4 and chemical compounds Cell. Stem Cell 2010 7, N 6:651–655.
[74] Hussein S. M., Batada N. N., Vuoristo S., Ching R. W., Autio R., Narva E., Ng S., Sourour M., Hamalainen R., Olsson C., Lundin K., Mikkola M.,Trokovic R., Peitz M., Brustle O., Bazett-Jones D. P., Alitalo K., Lahesmaa R., Nagy A., Otonkoski T. Copy number variation and selection during reprogramming to pluripotency Nature 2011 471, N 7336:58–62.
[75] Lister R., Pelizzola M., Kida Y. S., Hawkins R. D., Nery J. R., Hon G., Antosiewicz-Bourget J., O'Malley R., Castanon R., Klugman S., Downes M.,Yu R., Stewart R., Ren B., Thomson J. A., Evans R. M., Ecker J. R. Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells Nature 2011 471, N 7336:68–73.
[76] Mayshar Y., Ben-David U., Lavon N., Biancotti J. C., Yakir B., Clark A. T., Plath K., Lowry W. E., Benvenisty N. Identification and classification of chromosomal aberrations in human induced pluripotent stem cells Cell. Stem. Cell 2010 7, N 4 P. 521–531.
[77] Hanna J. H., Saha K., Jaenisch R. Pluripotency and cellular reprogramming: facts, hypotheses, unresolved issues Cell 2010 143, N 4:508–525.
[78] Kim K., Doi A., Wen B., Ng K., Zhao R., Cahan P., Kim J., Aryee M. J., Ji H., Ehrlich L. I., Yabuuchi A., Takeuchi A., Cunniff K. C., Hongguang H., McKinney-Freeman S., Naveiras O., Yoon T. J., Irizarry R. A., Jung N., Seita J., Hanna J., Murakami P., Jaenisch R., Weissleder R., Orkin S. H.,Weissman I. L., Feinberg A. P., Daley G. Q. Epigenetic memory in induced pluripotent stem cells Nature 2010 467, N 7313:285–290.
[79] Cohen D. E., Melton D. Turning straw into gold: directing cell fate for regenerative medicine Nat. Rev. Genet 2011 12, N 4:243–252.