INDUCED PLURIPOTENT STEM CELLS ipsc FOR GENE THERAPY

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1 IMiD, Wydawnictwo Aluna Developmental Period Medicine, 2013, XVII, PRACE POGLĄDOWE/REVIEW S ARTICLES Ilona Szabłowska-Gadomska 1,2, Agnieszka Górska 1, Maciej Małecki 1,2 INDUCED PLURIPOTENT STEM CELLS ipsc FOR GENE THERAPY INDUKOWALNE PLURIPOTENCJALNE KOMÓRKI MACIERZYSTE ipsc W TERAPII GENOWEJ 1 Department of Applied Pharmacy and Bioengineering, Medical University of Warsaw, Poland 2 Institute of Mother and Child, Warsaw, Poland Abstract The generation of autologous pluripotent stem cells by reprogramming and then their differentiation into any cell type is a very attractive prospect for biomedicine. Additionally, if it were possible to repair malfunctioning genes it would mean that there is hope for patients suffering from incurable diseases in which conventional treatment does not give satisfactory results. Data from animal models are promising. But there are still issues that must be solved, such as the low efficiency of the derivation of induced pluripotent cells, and most importantly, making sure that the techniques used both for reprogramming cells, as well as for gene therapy are safe. Key words: Gene Therapy, Induced Pluripotent Stem Cells, genetic vectors Streszczenie Otrzymywanie, w wyniku reprogramowania, autologicznych komórek pluripotencjalnych a później ich różnicowanie w dowolny typ komórek jest bardzo atrakcyjną perspektywą rozwoju biomedycyny. Jeśli dodatkowo możliwe byłoby naprawianie nieprawidłowo funkcjonujących genów oznaczałoby to nadzieję dla pacjentów cierpiących na nieuleczalne choroby, u których konwencjonalne leczenie nie przynosi zadowalających rezultatów. Wyniki badań na zwierzętach są obiecujące. Jednak nadal do rozwiązania pozostają kwestie niskiej efektywności metod otrzymywania indukowanych pluripotencjalnych komórek macierzystych i co najważniejsze bezpieczeństwa technik wykorzystywanych zarówno do reprogramowania komórek, jak też terapii genowej. Słowa kluczowe: pluripotencjalne komórki macierzyste, terapia genowa, wektory genetyczne DEV. PERIOD MED., 2013, XVII, 3, In regenerative medicine huge hopes have been set on stem cells. Many patients are still waiting for a breakthrough in their use. The work done by the team led by Yamanaka proved to be a milestone in this field due to the introduction of genes encoding merely four reprogramming factors. This made it possible to obtain pluripotent cells ips (Induced Pluripotent Stem cells ipsc) from somatic cells. Induced pluripotent stem cells bear a high resemblance to embryonic stem cells (ES cells) in terms of morphology, gene expression and the potential of differentiaton (1). In the past scientists claimed that the way cells develop/differentiate is unidirectional and irreversible. The experience of cell reprogramming by using the techniques of nuclear transplantation SCNT (Somatic Cell Nuclear Transfer), cell fusion and currently by means of reprogramming factors (according to Yamanaka s method) contradict such an assumption (2, 3) (Fig. 1). The possibility of obtaining pluripotent stem cells from almost any somatic cell (without raising ethical objections relating to the use of oocytes and embryonic cells) is an unquestionable advantage of the latest technology.

2 192 Ilona Szabłowska-Gadomska i wsp. ReprogrammingIReprogramowanie Oocyte/Oocyt Somatic cell/komórka somatyczna ES cell/zarodkowa komórka macierzysta SCNT ES cell/zarodkowa komórka macierzysta Heterocarion/Heterokarion or/lub Somatic cell/komórka somatyczna Cell fusion / Fuzja komórek Hybrids/ Hybrydy Reprogramming factors/ Czynniki Reprogramujące Somatic cell/ Komórka somatyczna by Yamanaka s method / metodą Yamanaki Induced Pluripotent Stem Cells ips Indukowana pluripotencjalna komórka macierzysta ips Fig. 1. Strategy of cell reprogrammining. Ryc. 1. Strategie reprogramowania komórek. The most valuable feature of pluripotent stem cells (including ips cells) is their ability to divide, self-renew, as well as the potential to differentiate into cells of three germ layers. The same properties are exhibited by node cells isolated from embryonic ICM (Inner Cell Mass) at the blastocyst stage. The attributes of these cells enable the production of all the body building tissues. A greater potential is exhibited only by totipotent cells (zygote) which, in addition to body building cells, are also able to create extra-embryonic structures. In order to derive ips cells, the latest techniques are used, which are applied, among other things, in gene therapy (Fig. 2). The introduction of nucleic acids encoding the reprogramming factors (in order to obtain pluripotent stem cells) can be done with different types of vectors. The most effective method, which is unfortunately burdened with the highest risk of oncogenesis and potential reactivation of transgenes during cell differentiation, is based on the use of retro and lentiviruses. In both systems amplification of transgenes often entails the integration of the virus vector into the genome of a cell undergoing the reprogramming process (the host cell). Using a lentivirus vector makes it possible to introduce genes into non-dividing cells (4). As far as clinical application is concerned, integration free methods that enable the amplification of the gene that is being introduced independent of the material located on the chromosomes of the host cell, are more secure. When introduced into cells, adenoviral vectors exist in episomal form and allow a high but transient expression level (4, 5). The methods for introducing genetic material into cells can also be classified into those based on viral and non-viral vectors. Amidst the latter, there are some interesting strategies based on PiggyBAC and minicircle plasmids (6). When considering methods of cell reprogramming, control of the expression of the genes introduced is a crucial aspect to be taken into account (particularly the way of gene silencing). The aforementioned PiggyBAC system (PB) is the mobile genetic element and due to transposase enzyme activity not only makes it possible to insert sequences but also to cut them (7). Removing the transgene can also take place through the Cre-loxP strategy, in which the use of Cre recombinase can cut the inserted sequences located between the loxp sequences (8). The strategy of recombinant/reprogramming proteins is the alternative to the above-mentioned techniques of gene introduction. In order to induce the endogenous expression of the genes associated with pluripotency,

3 Induced Pluripotent Stem Cells (ipsc) for gene therapy 193 Reprogramming factors/ Czynniki reprogramujace Differentiation/ Różnicowanie Reprogramming/ Reprogramowanie Endoderm cells/ Komórki endodermy Somatic cell/ Komórka somatyczna Mesoderm cells/ Komórki mezodermy plasmid/ plazmid Virus/ wirusy mrna Ectoderm cells/ Komórki ektodermy Reprogramming protein/ Białka reprogramujące Small molecules/ Małe związki Fig. 2. Manufacturing of ips cells. Ryc. 2. Otrzymywanie komórek ips. two research teams (Kim s and Ding s) introduced four protein transcription factors OCT4, SOX2, KLF4 and C-MYC with attached polyarginine tails into the cell (9, 10). The polyarginine domains associated with the proteins introduced make it easier to overcome the barrier membrane. This mechanism also applies to naturally occurring peptides rich in arginine and lysine, therefore these proteins are called CPP (Cell Penetrating Peptide) (11). However, all these methods exhibit a very low efficiency as opposed to strategies based on retroviruses, as well as lentiviruses and require multiple and time consuming tests. A different reprogramming strategy using synthetic mrna was proposed by Warren and his team. This method is as good as integration techniques in terms of efficiency (5, 6). However, it carries a risk of malignancy as a result of such factors as, for example, over expression of MYC introduced to function not only as a reprogramming factor but also as a proto-oncogene, known for its cell proliferation. (12). In the case of gene therapy the most commonly used vectors include promoters allowing the constitutive expression of genes, which significantly limits the regulation/ control of expression of the transgene transcription. Nowadays, there are successfully made attempts to use adjustable promoters of genes whose activity depends on, inter alia, the pharmacological stimulation of cells. The other promoters, such as tissue - specific ones, arouse interest as well. In particular, great hopes have been reposed in the use of these methods for ischemic disease treatment a field in which many positive results have been documented (13). As an example, ips cells may be generated from the skin cells of patients with neurodegenerative diseases and then used as material for gene therapy. Afterwards, they may be implanted into patients after the manipulations associated with the repair and after differenciation into the congruent type of cells (14) (Fig. 3). So far ips cells have successfully been derived from patients with diseases like Huntington s, Parkinson s, Duchenne dystrophy or Amyotrophic Lateral Sclerosis (ALS) (16). A lot of information is provided from animal testing. Succesful examples include a completed study of the model of sickle cell disease. Hanna et al. (17) obtained ips cells from mice cells which exhibited a genetic defect. These cells were then fixed and, after differentiation, implanted back into mice that showed improvement, afterwards. Gene therapy may be an alternative form of treatment for patient with diseases such as congenital or single gene disorders, or cancer, especially when pharmacological or surgical intervention does not give good results (4, 18). In

4 194 Ilona Szabłowska-Gadomska i wsp. Gene therapy/ Terapia genowa Normal pre-differentiated cells/ Komórki prawidłowe podróżnicowane Autologous transplantation ips normal cells/ ips komórki prawidłowe ips altered cells/ ips komórki nieprawidłowe Transplantacja autologiczna Norma pre-differentiated cells/ Komórki prawidłowe podróżnicowane Normal somatic cells/ Komórki somatyczne prawidłowe Altered somatic cells/ Komórki somatyczne nieprawidłowe ips normal cells/ ips komórki prawidłowe Patient/ Pacjent Fig. 3. Poten al applica ons of ips cells for biomedicine. Ryc. 3. Potencjalne wykorzystanie komórek ips w biomedycynie. the future, induced pluripotent stem cells in combination with gene therapy may enhance the progress in treatment. Future work should focus on research in improving the methods of obtaining ips cells, as well as gene therapy techniques in order to become more effective and safe for patients. REFERENCES 1. Takahashi K., Yamanaka S.: Induction of pluripo tent stem cells from mouse embryonic and adult fibro blast cultures by defined factors cell, 2006, 126, Ladewig J, Koch P, Brüstle O.: Leveling Waddington: the emergence of direct programming and the loss of cell fate hierarchies. Nat. Rev. Mol. Cell. Biol. 2013, Apr, 14(4), Yananaka S., Blau H.N.: Nuclear reprogramming to a pluripotent state by three approaches, Nature 10, 2010, Józkowicz A., Dulak J.: Nowe strategie wykorzystania wektorów plazmidowych i wirusowych w terapii genowej, Biotechnologia 3, 78, 2007, Gonzalez F., Boue S., Belmonte J.C.I.: Methods for making induced pluripotent stem cells: reprograming a la carte, Nature Reviews Genetics, 2011, Oh S., Lee C.K., Cho K.J., Lee K., Cho S., Hong S.: Technological Progress in Generation of Induced Pluripotent Stem Cells for Clinical Applications, The Scientific World Journal, 2012, Woltjen K., Michael I.P., Mohseni P., Desai R., Mileikovsky M., Hämäläinen R., Cowling R., Wang W., Liu P., Gertsenstein M., Kaji K., Sung H.K., Nagy A.: PiggyBac transposition reprograms fibroblasts to induced pluripotent stem cells, Nature, 2009, Apr 9, 458(7239), Soldner F., Hockemeyer D., Beard C., Gao Q., Bell G.W., Cook E.G., Hargus G., Blak A., Cooper O., Mitalipova M., Isacson O., Jaenisch R.: Parkinson s Disease Patient-Derived Induced Pluripotent Stem Cells Free of Viral Reprogramming Factors. Cell, 2009, 136, Zhou H., Wu S., Joo J.Y., Zhu S., Han D.W., Lin T., Trauger S., Bien G., Yao S., Zhu Y., Siuzdak G., Schöler H.R., Duan L., Ding S.: Generation of induced pluripotent stem cells using recombinant proteins, Cell Stem Cell, 2009, May 8, 4(5), Kim J.B., Greber B., Araúzo-Bravo M.J., Meyer J., Park K.I., Zaehres H., Schöler H.R.: Direct reprogramming of human neural stem cells by OCT4. Nature, 2009, 461(7264), Ziegler A., Nervi P., Dürrenberger M., Seelig J.: The Cationic Cell-Penetrating Peptide CPPTAT Derived from the HIV-1 Protein TAT Is Rapidly Transported into Living Fibroblasts: Optical, Biophysical, and Metabolic Evidence. Biochemistry. 2005, 44 (1),

5 Induced Pluripotent Stem Cells (ipsc) for gene therapy Bernard S., Eilers M.: Control of cell proliferation and growth by Myc proteins, Results Probl. Cell Differ, 2006, 42, Malecki M., Kolsut P., Proczka R.: Angiogenic and antyangiogenic gene therapy. Gene Therapy 2005, 12, Gibson S., Gao G., McDonalds K., Shen S.: Progress on stem cell research towards the treatment of Parkinson s disease, Stem Cell Research & Therapy, 2012, Arnold A., Naaldijk Y., Fabian C., Wirth H., Binder H., Nikkhah G., Armstrong L., Stolzing A.: Reprogramming of Human Huntington Fibroblasts Using mrna, ISRN Cell Biology, 2012, Archacka K., Grabowska I., Ciemerych M.: Indukowane Komórki Pluripotentne Nadzieje, Obawy i Perspektywy, Postępy Biologii Komórki, 2010, Hanna J., Wernig M., Markoulaki S., Sun C., Meissner A., Cassady J., Beard C., Brambink T., Wu L., Townes T., Jaenisch R.: Treatment of Sickle Cell Anemia Mouse Model with ips Cells Generated from Autologous Skin, Science 21, vol 318, 2007, Małecki M., Janik P.: Terapia genowa w klinice, Współczesna Onkologia, 2004, vol. 8, 3, Author s contributions/wkład Autorów According to the order of the Authorship/Według kolejności Conflicts of interest/konflikt interesu The Authors declare no conflict of interest. Autorzy pracy nie zgłaszają konfliktu interesów. Received/Nadesłano: r. Accepted/Zaakceptowano: r. Published online/dostępne online Address for correspondence: Maciej Małecki Department of Applied Pharmacy and Bioengineering, Medical University of Warsaw Banacha 1, Warsaw, Poland tel maciej.malecki@wum.edu.pl