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1162 ParT TEN Prevention and Therapy of Immunological Diseases

Codon#: 1 2 3 4 5 6 7 8 9 Gene Sequence-specific
(1) Wild-type 5’-ATG CCTTGA AATTCG GGGCGA TTGACC-3’ endonuclease Mutation
Gene 3’-TAC GGAACT TTAAGC CCCGCT AACTGG-5’ Promoter
1 2 3 4 5
(2) Mutant 5’-ATG CCTTGA AAT ACG GGGCGA TTGACC-3’
Gene 3’-TAC GGAACT TTA TGC CCC GCTAAC TGG-5’
1 2 34 5 pA Homology arm flanked,
(3) Repair template 5’-GA AAT TCG GGGC-3’ codon-optimized cDNA/pA
Donor
(4) Corrected 5’-ATG CCTTGA AAT TCG GGG CGATTG ACC-3’
Gene 3’-TAC GGAACT TTA AGC CCC GCTAAC TGG-5’
FIG 85.3 Site-Specific Gene Repair by Homologous Recom- Inserted cDNA transcribed
from endogenous promoter
bination (HR). In this example, instead of the wild-type gene Mutation
sequence (1), a patient’s gene (2) has a mutation from a base-pair Promoter 1 2 3 45 pA 1 2 3
substitution of an A for a T at the start of the 5th codon (red).
An artificial donor template (3), here as a single-stranded AAAAA
deoxyoligonucleotide of 12 bases in length (green), is provided FIG 85.4 Site-Specific Insertion of a cDNA to Override Down-
with the correct base present at the site of the patient’s mutation Stream Gene Mutations. A prototypical gene is shown with 5
(blue). If the donor template is used to repair a double-stranded exons (yellow boxes 1–5) and an upstream promoter (red box).
DNA break induced near the mutation by a site-specific endo- A mutation in exon 2 (black X) inactivates the gene. A sequence-
nuclease (red arrow), sequences from the donor (green) will be specific endonuclease (red arrow) is designed to introduce a
incorporated into the patient’s gene (4), introducing the normal double-stranded DNA break, in this case in the 5′ untranslated
corrective base pair (blue). region of the gene (blue and green lines). A cDNA molecule
contains the contiguous exons of the gene (orange 1–5), codon-
optimized to increase expression and to eliminate homology
with the endogenous exons to eliminate illegitimate recombination
transcriptional factors such as CTLA-4, or STAT3 Gain of Function events, and a polyadenylation signal (pA) to terminate transcrip-
alleles, a transcriptional repressor of fetal hemoglobin, etc. There tion. The cDNA is flanked by the sequences from the endonucle-
have been clinical trials for patients with HIV infection in which ase cleavage site (blue and green lines). The donor can be inserted
the gene for CCR5 HIV co-receptor was disrupted using a zinc into the nuclease target site by homologous recombination,
finger nuclease (ZFN) to make a double stranded DNA break placing the cDNA under transcriptional control of the endogenous
while NHEJ was allowed to repair the break, leading to indels gene promoter; the cDNA transcript (red arrow) would override
that inactivated the gene and co-receptor expression. 27 any downstream mutations in the gene.
HR is a more precise repair mechanism that normally uses a
copied sister chromosome or the other homologue as a template
to repair the break; sequences of the template are copied into
the repair site and if there are differences, the template acts as a Clustered, Regularly Interspaced, Short Palindromic Repeats
donor for the new sequences. For gene correction, an artificial (CRISPRs) that allow the introduction of a double-stranded
donor template is provided to the cells to instruct the introduc- DNA break at unique sites in the mammalian genome with high
tion of the intended sequence changes (Fig. 85.3). Beyond its specificity. Current methods introduce the nuclease into HSCs
use for modification of single base pairs, as illustrated here, in a method where it will only be present transiently, such as
HR can be used to introduce whole gene sequences into the electroporation of in vitro transcribed messenger RNA encoding
target site by flanking a gene cassette with “homology arms” the nuclease proteins. The homologous donor template is
that consist of the DNA sequences homologous to the target site introduced as either a short (e.g. 50–100 bases) single-stranded
(Fig. 85.4). deoxy-oligonucleotide that is co-electroporated with the nuclease,
Although HR has been used to introduce genes into cells, or as a longer sequence carried by a viral vector that does not
such as murine embryonic stem cells to make gene knockout integrate into the target cell chromosome, including adeno-
and knockin mice, it is generally a low-frequency event (occurring associated virus (AAV) or an integrase-defective lentiviral vector
6
4
in 1/10 –1/10 cells) and requires the use of selectable markers (IDLV). The development and application of this technology
to isolate the rare desired recombinant. Whereas cloning and has evolved at an incredibly rapid pace and is approaching the
expansion of murine (and human) embryonic stem cells can be levels of efficiency needed for clinical applications.
done to produce populations of the rare recombinant cells, this One caveat is that potential off-target activity of the nucleases
is not possible with primary HSCs, which cannot be expanded could cause genotoxicity from either disruption of unintended
to any great degree from single cells. Methods achieving high genes or even introduction of chromosomal translocations
efficiency of gene modification with low cytotoxicity in large between two cut sites produced simultaneously, e.g., one on-target
numbers of primary stem cells are needed for clinical applications and one off-target. Current studies are assessing the consequences
to autologous HSCT. The major breakthrough in this area comes of this potential genotoxicity in human HSCs, while ongoing
from the observation that HR is vastly more frequent when a work is seeking to improve the specificity of the nucleases to
double-stranded DNA break is introduced close to the target eliminate or greatly minimize off-target activity.
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site; then, the repair donor molecule can be used at efficiencies Gene correction will have several advantages over gene
in the range ~1–50% of treated cells. addition. It would avoid the potential problems from randomly
Several classes of designer site-specific endonucleases have inserting vectors that may disrupt or over-stimulate adjacent
been derived, including ZFN, introduced above; Transcription cellular genes, as discussed above for the retroviral vectors.
Activator-Like Effector Nucleases (TALENs); and more recently Crucially, correcting the endogenous gene keeps its expression