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452 Part V: Therapeutic Principles Chapter 30: Regenerative Medicine: Multipotential Cell Therapy for Tissue Repair 453

chemicals, or growth factors. Current protocols for differentiation activator-like effector nucleases (TALENs), and clustered regularly
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of pluripotent stem cells to beta cells follow a five-stage procedure that interspaced short palindromic repeats (CRISPRs)/Cas9 system.
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recapitulates the embryonic stages of development. Only the first four Each class of reagent has been used for stem and progenitor cell
stages have been carried out successfully in vitro. The fifth stage—which genome modification with ZFNS, which is, to date, the first to enter clin-
involves maturation to glucose-responsive, insulin-secreting beta cells ical application. Human ESC engineering with ZFNs was first used to
and other islet cells—until recently could only be carried out by implan- target the HUES-3 and HUES-1 cell lines with ZFNs designed for inac-
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tation in vivo. A long-awaited directed differentiation of insulin- tivation of the CCR5 gene, a coreceptor for HIV entry to a cell. ZFNs
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producing cells from hESCs has been accomplished; this fully defined and a donor sequence containing green fluorescent protein (GFP) were
ex vivo technology is immediately relevant. 153 introduced into exon 3 of the CCR5 gene and approximately 5 percent
Also nonendocrine cells within the pancreas have been found to rates of targeted integration were observed. Importantly, the cells main-
transdifferentiate or reprogram to a beta cell fate upon their being tained their pluripotency and ability to self-renew. This study estab-
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induced with the three-pancreatic-gene cocktail (Pdx1, Ngn3, and lished a precedent for inserting genes of interest into a specified spot in
MafA). Another source for adult cell reprogramming to beta cells has the ESC genome via HR. Others extended this to allow for gene addition,
been described from glucagon-producing alpha cells. A study showed a placement of an inducible expression cassette at the so-called safe har-
that overexpression of Pax4 (a gene responsible for specifying endo- bor locus AAVS1. Using ZFNs for the first exon of the PPP1R12C gene
crine fate) in the alpha cells was able to force them to become beta-like on chromosome 19, a “standalone” expression cassette was introduced
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cells. In another study, it was observed that near-complete ablation containing a promoter, puromycin gene, and a polyadenylation signal
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of beta cells forced regeneration of beta cells from former alpha cells. (or gene trap targeting vector) containing a splice acceptor-2A-puromy-
However such in vivo studies have not been established in humans or cin gene that relied on proper targeting and splicing with the first exon
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other primates. of the PPP1R12C gene. As such, gene targeting at the AAV locus allows
for placement of a gene with a promoter that drives the desired level
GENE EDITED MULTIPOTENTIAL CELLS of expression or is controlled by the native PPP1R12C promoter that
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is constitutively active. In another study, employing the safe harbor
The ability to correct defective cells or give them enhanced properties strategy did not appear to alter the pluripotent nature of ESCs. 161–163 The
(e.g., antitumor effects) represents a novel approach to transplantation ability to modify genes in pluripotent target cells is important to disease
medicine and sets the stage for individualized therapies. Two options modeling in vitro, which has become a new foundation for the accel-
exist for this strategy: provision of functional copies of a gene deliv- eration of translational research. Although these studies established the
ered by a viral or nonviral gene transfer system or in situ correction ability to modify ESCs at a site-specific and “safe harbor” locus, wide-
of the disease-causing sequence. Several major studies have used clin- spread use is limited by the relatively small number of approved ESC
ically employed viral transgenesis of hematopoietic stem cells (HSCs). lines and the even smaller number of disease-specific ones.
In 2010, a γ-retroviral vector was used to deliver the complementary As an elegant solution to address the potential paucity of disease-
DNA for the IL2RG gene to CD34+ progenitors from patients with specific stem cells and to remove the potential for variability between
X-linked severe combined immunodeficiency (SCID-X1). Normaliza- stem cell lines, ZFNs have been used to generate isogenic control and
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tion of the immune system occurred in most patients; however, four Parkinson disease (PD) cell lines. This work centered on engineering
patients developed acute T-cell leukemia from promiscuous LMO2 the A53T or E46K PD mutations into disease-free ESCs or repair-
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oncogene activation by the viral vector. To mitigate the potential for ing the mutation in PD patient-derived iPSCs. In this way they miti-
viral elements to dysregulate endogenous gene expression, investiga- gated the effects of the numerous genetic differences and modifiers that
tors have used self-inactivating lentiviral vectors for the gene therapy exist between individuals and ESC and iPSC clones.
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of X-linked adrenoleukodystrophy, metachromatic leukodystrophy, Toward realizing the therapeutic potential of stem cells, investi-
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and Wiskott-Aldrich syndrome, in gene therapy trials using hemato- gators derived a fibroblast cell line from a humanized mouse model of
poietic progenitors. sickle cell anemia; reprogrammed these cells into iPSCs; performed
Despite this, the integrating nature of viral vectors, with a prefer- gene correction using a plasmid donor; differentiated the cells into
ence for transcriptionally active areas, makes more precise gene target- hematopoietic progenitor cells; and transplanted them into sickle cell
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ing a highly desirable goal. Such precision can be achieved with genome mice to reconstitute normal erythropoiesis. Proof of principle for a
editing nucleases that are rationally designed and with engineered pro- similar strategy using human cells was demonstrated using ZFNs to
teins that have the unifying characteristic of recognizing and contacting correct the sickle cell mutation in iPSCs that were subsequently differ-
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a unique sequence of DNA. Most studies have tethered these proteins to entiated into cells of the erythroid lineage. Numerous studies using
nuclease domains or used their inherent ability to cut DNA. Once the ZFNs, TALENs, and CRISPR/Cas9 have shown the ability to correct
DNA is broken, two predominant repair pathways have been used for disease-causing mutations in iPSCs or in primary cells that are subse-
therapeutic genome engineering: nonhomologous end-joining (NHEJ) quently differentiated into iPSCs. A major limitation for these strategies
and homologous recombination (HR). NHEJ is an error-prone path- for hematologic disorders is the poor and/or absent ability of iPSCs to
way that, in the absence of a donor template, repairs the DNA break form from true blood progenitors ex vivo that are capable of reconstitut-
in a way that can cause small insertions or deletions (“indels”) that can ing a functional circulatory system. However, the most streamlined path
permanently disrupt coding DNA sequences. Gene repair relies on the to translational use is likely to be direct modification of a patient’s own
error-free HR pathway. In gene repair, the inclusion of an exogenous HSCs. To date only two reports document the ability to mediate HR
single- or double-stranded DNA donor template that contains homol- in HSCs. In 2007, maximal rates of 0.11 percent gene targeting at the
ogous sequences to the target site avoids disruption. In response to a CCR5 locus utilizing ZFNs and a donor containing GFP or a puromy-
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double-strand break (DSB), the donor template acts as the template cin resistance gene were reached. Subsequently, optimized conditions
for repair and allows for the precise and permanent insertion of user- involving ZFNs delivered as mRNA and the donor construct delivered
defined sequences at the target locus. Both repair pathways can be used on an integrase-deficient lentiviral (IDLV) cassette were used to cor-
therapeutically. The major candidates employed for DNA cleavage are rect the IL2RG gene from an individual with SCID-X1 and observed
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the meganucleases (MNs), zinc finger nucleases (ZFNs), transcription multilineage repopulation in transplanted mice. This specialized

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