Page 1744 - Hematology_ Basic Principles and Practice ( PDFDrive )

C H A P T E R 98

PRINCIPLES OF CELL-BASED GENETIC THERAPIES

David A. Williams

The use of gene transfer to treat human diseases has now been manipulation has been the most difficult to consistently meet in
demonstrated to be efficacious in a limited number of instances. human applications using current gene transfer technology.
Proof-of-principle successes in several monogenic diseases—both Since the early development of virus vectors, blood-forming cells
hematologic and nonhematologic—have been published and widely have been used as one optimal target for ex vivo gene transfer studies.
publicized in the past decade. The previous occurrence of serious For this purpose, hematopoietic stem and progenitor cells (HPSCs)
adverse events in some trials related to insertional mutagenesis has are isolated, manipulated in the laboratory, and administered back to
stimulated rapid development of safer vector systems. This chapter the patient. The advantages of these cells as targets of gene transfer
discusses the basic biology of vector systems applicable to blood are multiple. First, all blood cells are derived from a common progeni-
diseases, the details of the application of gene therapy to blood dis- tor cell, the hematopoietic stem cell (HSC), which is both long lived
eases using specific trials as examples of this technology, and the in vivo and capable of significant self-renewal. The latter capacity and
modifications in vector systems driven by clinical experience that the pluripotency of HSCs is exploited to amplify the genetically
predict future trials. The chapter also discusses the evolving field of manipulated cells into large cell numbers of multiple blood lineages
somatic cell reprogramming and genome editing that may impact derived from the genetically altered cells in vivo. There is a long and
clinical applications in the future. successful experience in obtaining these stem cells from the bone
marrow (BM) and peripheral and umbilical cord blood. There is
HEMATOLOGIC DISEASES, CELLULAR TARGETS, AND extensive experience in the use of HSCs in the clinical setting for
transplantation, and there is experience in purification of these cells
THE BASIS FOR GENETIC THERAPIES and limited knowledge of the requirements for ex vivo manipulation
of the cells. In addition, the experience of HSC transplantation
Gene therapy is defined as the introduction of new genetic material (HSCT) has defined a variety of genetic diseases in which the phe-
into the cells of an organism for therapeutic purposes. Broadly speak- notype can be altered by the successful engraftment of normal allo-
ing, two types of gene therapy can be envisioned. The introduction geneic donor cells. Finally, the blood system is involved as a major
of genetic material into germ cells such that the new DNA can be dose-limiting organ in cancer therapies and both a target and an
expected to be passed into the gene pool. This is termed germline gene effector organ in immune reactions, providing a large group of dis-
therapy and is currently banned in the United States and around the eases that could theoretically be approached using gene transfer
world. The potential for newer methods of genome engineering to technology. As noted earlier, there are already a large number of
be utilized in clinical applications (discussed later) has led to more monogenic diseases of the blood extensively characterized, with more
recent calls for extending this restriction. In contrast, introduction of being defined at the molecular level on a regular basis as whole-exome
new genetic material into specialized cells of the body with no risk and whole-genome sequencing is being applied to rare disease phe-
of the new genetic material being passed onto subsequent generations notypes. In addition to HSC targets, another application of gene
is termed somatic gene therapy. The ultimate goal of gene therapy is transfer technology exploits the experience in adoptive T-cell immu-
to correct the targeted genetic disease by replacement of the defective notherapy. In this application, T cells (and less well developed to this
gene in situ. Such gene replacement could be envisioned via a process point, other immune effector cells) are modified ex vivo in an attempt
termed homologous recombination. Homologous recombination in to enhance potency and specificity. This application of gene transfer
mammalian cells is widely practiced in laboratories but up to now technology will not be reviewed here.
has been relatively inefficient, although newer technology may The field of gene therapy is rapidly evolving. Successes of “proof-
overcome some of the previous limitations to efficient genome of-principle” small trials have demonstrated the utility of gene transfer
editing. Advantages of this approach would include a reduction in approach in a sizable number of patients but in a limited number of
the risk of inadvertent disruption or dysregulation of expression of a diseases. The technology itself is quickly evolving in response to new
critical gene sequence and regulated (appropriate level and distribu- understanding of viruses, the regulation of gene expression, and gene
tion) expression of the normal (replaced) gene. However, the frequency editing. The application of gene transfer technology to HSC gene
of homologous recombination (in contrast to random chromosomal therapy has been made possible by exploitation of viruses that have
integration) in mammalian cells and primary tissues makes therapeu- evolved the capacity to efficiently and precisely insert viral genomes
tic use of homologous recombination somewhat impractical at this into cellular chromosomes of infected cells. The field has taken more
point. Methods to effect homologous recombination have improved than 30 years to evolve to its current state of clinical application.
in the past 5 years and may make this goal attainable in the future. Although this might be viewed as a slow pace, in reality, this time
The requirements for successful application of our current gene frame parallels the development of many other novel therapies. This
transfer technology for treatment of human diseases include knowl- developmental phase also reflects the complexities of the biologic
edge of the abnormal gene sequence responsible for the disease systems involved and the caution required in moving forward in the
phenotype and the availability of the corresponding normal gene face of serious adverse events seen in early safety trials. It is indeed
sequence that can be packaged into current vector backbones for an exciting time with respect to the clinical application of gene
efficient recombinant virus production. In addition, the cells respon- transfer technology in human diseases.
sible for the disease phenotype must be identified and accessible for
genetic manipulation. Finally, a means of introducing and expressing
the correct gene sequence in cells such that the disease phenotype can VECTOR SYSTEMS
be reversed is needed. Although effectively accomplished nearly three
decades ago in murine studies, this latter requirement encompassing The initial impetus to develop gene transfer for human studies
both in vivo administration of DNA sequences and ex vivo cell derived from the exploitation of oncoretroviruses, mainly murine

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