Page 1752 - Hematology_ Basic Principles and Practice ( PDFDrive )

Chapter 98 Principles of Cell-Based Genetic Therapies 1557

from inner cell mass-derived ES cells. Takahashi et al reasoned that Finally, Naldini and colleagues have demonstrated that targeted inser-
forcing the expression of ES cell-specific genes, particularly transcrip- tion into two genomic sites (IL2RG and CCR5) using zinc-finger
tion factors in somatic cells, might induce (reprogram) somatic cells technology leads to no detectable alteration in the expression of
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to take on the properties of ES cells, much like factors present in nearby genes and sustained expression of the transgene cargo. The
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oocytes can reprogram somatic nuclei in mammals. By systematic frequency of directed site-specific integration in primary fibroblasts
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screening experiments, four factors, including some known to be was as high as 10%. In addition, elegant work by Sadelain and
involved in the process of self-renewal (Oct3/4, Sox2), and others colleagues has demonstrated the feasibility of screening large numbers
associated with transformation and maintenance of ES cell pluripo- of iPS-derived clones for both integration in operationally defined
tency (c-Myc, Klf 4), were identified as sufficient to achieve repro- safe harbors and for clinically relevant transgene expression using
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gramming. The resultant cells, termed iPS cells, exhibited key features vectors that express globin genes. Thus, this approach offers exciting
of ES cells, including the fulfillment of stringent pluripotency future applications in treating a variety of human diseases.
requirements. This work was subsequently confirmed and expanded
upon by other groups describing the application of this technology
to human cells. Proof-of-concept for the utility of iPS cells in regen- Site-Directed Homologous Recombination to
erative medicine of inherited blood disorders was recently provided Correct Gene Mutations
when iPS cells were generated from tail tip fibroblasts in a humanized
sickle cell anemia mouse model. The genetic sickle hemoglobin defect A long-standing but unrealized goal of genetic therapy has been
of the iPS cells was corrected in vitro via homologous recombination. homologous recombination to affect replacement of abnormal
Using established protocols for the differentiation of hematopoietic disease-causing gene mutations. Although human ES cells theoreti-
progenitors from ES cells, hematopoietic cells capable of reconstitut- cally provide an ideal target for such correction, gene targeting in
ing lethally irradiated recipient mice were generated. these cells has proven difficult. Zinc-finger nuclease (ZFN)-mediated
Transplantation of these cells into irradiated recipients resulted in DS breaks allow high-efficiency site-specific homologous recombina-
robust engraftment and amelioration of the sickle cell phenotype in tion and has been used to target a number of genes in human cells.
transplant recipients. Reprogramming of a panel of disease-specific ZFNs are generated by fusing the FokI nuclease domain to a DNA
human iPS cells, including patients with blood diseases, has been recognition domain composed of engineered C 2 H 2 zinc finger motifs
demonstrated. Human iPS cell lines shared defining features with that specify the genomic DNA binding site for the chimeric protein.
human ES cells, including morphology, proliferation, feeder depen- Compared with standard homologous recombination vectors, one
dence, surface markers, gene expression, promoter and telomerase advantage of ZFN is the relatively short stretch of homology needed
activities, in vivo differentiation, and teratoma formation. In analogy (500 bp vs. 10–12 kb) to mediate genomic targeting. Binding of two
to the murine system, the reprogramming viruses are strongly silenced fusion proteins to cognate DNA allows dimerization of the nuclease,
in human iPS cells, indicating that the maintenance of pluripotency leading to generation of a DS DNA break. When donor DNA with
does not depend on continuous transgene expression. Enforced homology to sequence flanking the DS break is present, repair occurs
transgene expression appears to initiate a sequence of stochastic events with incorporation of the incoming DNA sequence. This system has
over several days that eventually induces a small fraction of cells been successfully used to target genes in multiple species and has
(0.001–0.5% of cells) to acquire a stable pluripotent state. During recently been demonstrated in both human ES cells and iPS cells to
direct reprogramming, gradual changes lead to a stable epigenetic effectively target both expressed and nonexpressed genes with a fre-
state that is indistinguishable from inner cell mass-derived ES cells. quency of 1% to 20% and has been termed genome editing. Because
For example, the Dnmt3a and Dnmt3b methyltransferases become the development of a DNA DS break is a prerequisite to ZFN-
activated and silence the viral transgenes as endogenous pluripotency mediated gene editing and such breaks induce both p53 and HDR,
factors are transcriptionally reactivated. Human iPS cell lines thus this approach, although attractive, could also have significant “off-
represent a novel stem cell population that can be studied with regard target” effects. In addition, as efficiency improves, direct targeting in
to normal and pathologic tissue formation in vitro, enabling disease HSCs is possible and a trial is currently under development to utilize
investigation, drug development, and a platform for producing ZFN-mediated deletion of BCL11A, which repressed fetal hemoglo-
autologous cell therapies that avoid immune rejection. Moreover, the bin expression for the treatment of β-thalassemia. 25
creation of iPS cells allows the correction of genetic defects and Other systems that seek to target specific genetic loci have also
exhaustive molecular characterization at the clonal level before tissue been described and are at different levels of development. As with
reconstruction. While genetic transduction with exogenous genes, ZFN nucleases, these approaches use cellular DNA repair mechanisms
particularly oncogenes such as c-MYC and KLF4, and the use of to introduce exogenous DNA sequences into the chromosome. Thus,
integrating retroviral delivery systems are clear handicaps of this methods to increase the efficiency of targeting by introducing DNA
technology with regard to future clinical translation, newer methods DS breaks as well as several other methods have exploited endonucle-
have utilized nonintegrating vectors and even mRNA transduction ases that target rare DNA sequences to establish these DS breaks.
to generate iPS. However, to date, robust reconstitution of murine Alternative methods in development include meganucleases and
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hematopoiesis has not been consistently demonstrated, and there are transcription activator-like effector nucleases. Even more recently,
no successful reports of in vivo reconstitution of human hematopoi- bacterial immunity-related clustered regularly interspaced short pal-
esis in model systems. These all represent significant barriers to the indromic repeats have shown higher efficiencies of genome editing
translation of this powerful technology into human therapies. by expressing the Cas9 protein and guide RNAs to target the mam-
One utility of targeting iPS cells for genetic therapies is that these malian genome at specific locations. Challenges of all of these
cells can be cloned and expanded, allowing analysis of vector insertion approaches include efficiency of targeting in primary cells and poten-
sites before clinical use. In addition, reprogramming has now been tial off-target effects on the genome, and will likely also depend on
accomplished, albeit at lower efficiencies using both nonintegrating technologic advances that allow ex vivo HSC cloning and large-scale
vectors and protein transduction. Reprogramming has also now been expansion. At present, the use of ES or iPS cells is clearly amendable
accomplished with the expression of fewer transcription factors, most to this approach, but as noted earlier, use of these cells to derive
notably without c-Myc. Several laboratories have demonstrated that transplantable HSCs is a major impediment to clinical utilization.
targeting specific loci is associated with limited or no adverse effects
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on expression of neighboring genes (reviewed by Sadelain et al ). For
example, targeting of the AAVS1 locus located on chromosome 19 FUTURE DIRECTIONS
has been well characterized. This locus encodes the PPP1R1C gene
that is ubiquitously expressed. Insertion at this site appears to provide The use of gene transfer to treat human diseases has now been suc-
a “safe harbor” with respect to genotoxicity and allows stable and cessful in several diseases. Compared with allogeneic BMT, ex vivo
long-term expression of transgenes in human embryonic stem cells. gene therapy using autologous cells obviates the need to search for