Page 1751 - Hematology_ Basic Principles and Practice ( PDFDrive )

1556 Part IX Cell-Based Therapies

insertional mutagenesis was initially perceived by many investigators developed distinct mechanisms of integrase-dependent integration
to be very low. The lack of efficacy in preclinical models using human that may have an impact on the mutagenic potential of recombinant
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hematopoietic stem and progenitor (CD34 ) cells also shifted empha- retroviral vectors. If one considers the possibility of integrating vectors
sis away from the risks of insertional mutagenesis. However, soon upregulating oncogene expression via either read-through transcrip-
after the publication detailing the efficacy of the Paris-based SCID-X1 tion or enhancer effects on the endogenous promoter, then γ-retroviral
trial, work emerged from the group of Baum and colleagues that vectors could be considered as potentially more mutagenic than
would reestablish the importance of insertional mutagenesis as a lentiviral vectors in this context because of their preference for inte-
significant risk factor in the retroviral-mediated genetic correction of gration near the TSS. Conversely, preferential integration within the
hematopoietic cells and that ultimately predicted the appearance of body of the primary transcript may result in lentiviral vectors having
leukemia in the SCID, CGD, and WAS gene therapy trials described a higher probability of interrupting, for example, tumor suppressor
earlier. Baum demonstrated for the first time that a replication- gene expression, or as noted earlier in the treatment of one patient
incompetent retroviral vector backbone designed for gene therapy with thalassemia in altering normal gene splicing. Progress has been
applications could cause cellular transformation via insertional made in the development of model systems to functionally evaluate
mutagenesis in the context of a transplant model of transduced the relative mutagenic potential of different vector systems. However,
murine HSCs. In this and a subsequent study, it was found that a the model systems developed to date have a clear preference to detect
single retroviral insertion in the vicinity of the ecotropic viral integra- mutagenesis mediated via upregulation of oncogene transcription. It
tion site 1 (Evi1) gene or the related PR domain-containing 16 is not clear whether this is a reflection of tumor suppressor gene
(PRDM16) gene resulted in their overexpression, likely because of inactivation being inconsequential as a mechanism of insertional
the influence of the LTR viral enhancer element, and was sufficient mutagenesis or is a result of bias within the model system. Clearly,
to initiate a cascade of events resulting in leukemic transformation in the preliminary results from the β-thalassemia trial described earlier
vivo. Furthermore, a high copy number infection of murine BM with demonstrate that lentiviral vectors may mediate insertional mutagen-
recombinant retroviral vectors was able to facilitate combinatorial esis via alternate mechanisms.
hits, which caused leukemogenesis. The pattern of cellular genes that As noted earlier, other related retroviral vector systems have also
combine to promote cellular transformation demonstrated a signifi- been shown to have an integration pattern that is distinct from
cant overlap with those that are deregulated in experiments that used γ-retroviral vectors, and as such may represent a safer vector configu-
RCR vectors to provoke the development of leukemia. Although ration. Recombinant foamy virus vectors do not preferentially inte-
murine HSCs likely represent a more readily transformed target than grate within genes, and their integration pattern does not significantly
their human counterparts, these studies formally established the correlate with actively transcribed genes. Likewise, avian sarcoma
mutagenic potential of recombinant retroviral vectors intended for leukosis (α-retrovirus) vectors do not favor gene-rich regions or TSS
gene therapy applications. as preferred integration sites. However, these novel vectors systems
Baum’s group then made the seminal observation that at low copy have not been as well characterized as γ-retroviral or lentiviral vectors
numbers, retroviral-transduced murine HSCs are selectively expanded with regards to safety and efficacy, and have not yet been translated
during transplant dependent upon proviral insertion sites. These to clinical use in the near future.
nonmalignant dominant clones are enriched for proviral integration
sites in the locale of genes encoding signal transduction molecules
and growth-promoting genes. Analysis of mRNA expression levels in
these clones revealed that the proviral insertion did indeed alter RECENT MODIFICATIONS OF VECTOR SYSTEMS BASED
transcriptional regulation of genes proximal to the integration site ON CLINICAL EXPERIENCE
and led to the hypothesis that this was a powerful method to identify
proengraftment genes through positive selection. These observations New Cell Targets in Genetic Engineering
were found to have direct translational relevance in the gene therapy
trial for CGD where the nonmalignant expansion of dominant ret- Despite recent advances in improving ex vivo manipulation of HSCs
roviral transduced clones in two patients was found to correlate with for the purpose of gene transfer, an ongoing limitation of this tech-
insertional upregulation of growth-promoting genes. Subsequent nology is the loss of engraftment potential of manipulated cells. This
experience with γ-retroviruses used in the clinical trial for WAS is dramatically emphasized in genetic diseases such as Fanconi anemia,
confirmed that insertional leukemogenesis in the SCID-X1 trial was in which the disease process itself leads to a marked reduction in HSC
not a disease-specific side effect. Multiple children in the WAS trial targets and, in addition, in HSCs, which appear to have increased
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have developed T-cell leukemia. Thus far, leukemias associated with susceptibility to in vitro stress. An exciting area of recent research
replication-incompetent retrovirus vectors are associated with inser- advances that may in the future address this issue and also allow
tional activation of known proto-oncogenes by viral promoter and prescreening at the molecular level of insertions to determine safety
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enhancer sequences (reviewed by Kohn et al ). is the use of embryonic stem (ES) or induced pluripotent stem (iPS;
Wild-type and recombinant retroviral vectors (including α, γ, next paragraph) cells. These cells offer exciting possibilities for study-
spuma, and lenti) integrate into the host genome in a semi-random ing mechanisms of pluripotency; establishing models for disease-
manner and demonstrate insertion site biases that are dependent on specific investigations; and enabling future applications in genetic
the accessibility of the insertion site in the target cell and variations and cellular therapies, including tissue engineering for regenerative
in the viral integrase enzyme that depend on retroviral genus. medicine. ES cells can be propagated and expanded in vitro, making
γ-retroviruses such as MLV have been shown to exert a clear prefer- them amenable to manipulations, including the genetic correction of
ence for integration in the region immediately surrounding the TSS molecular defects. ES cells can be differentiated into a number of cells
of actively transcribed genes. Although lentiviral vectors also demon- resembling mature cell types in vitro. In theory, cell therapy using
strate a preference to integrate within the loci of actively transcribed donor cells of the same genetic constitution as the recipient may avoid
genes, their integration profile favors sites that are downstream of the the issues related to the immune barrier of allogeneic transplantation.
TSS within the body of the primary transcript. One of the key ethical and practical barriers to applying ES cell
Using viral chimeras, it has been shown that incorporation of research to human diseases is the need for obtaining human oocytes
MLV integrase into an HIV-1–based vector alters the integration or embryos for the in vitro generation of ES cell lines.
pattern of the lentivirus to more closely resemble that associated with A key recent finding with potential applications in cell and gene
a γ-retroviral vector. It is possible that this phenomenon results from therapy has been the observation that many somatic tissues can be
the binding of the MLV integrase with cell type-specific transcription “reprogrammed” into pluripotent stem cells with characteristics of ES
factors, resulting in the recruitment of the preintegration complex to cells, termed iPS cells, and direct reprogramming of differentiated
the promoter and enhancer region of actively transcribed genes. This somatic cells by gene transfer of a small number of defined transcrip-
work clearly demonstrates that γ-retroviral and lentiviral vectors have tion factors has been shown to yield cells that are indistinguishable