Page 1745 - Hematology_ Basic Principles and Practice ( PDFDrive )

1550 Part IX Cell-Based Therapies

γ-retroviruses, as vectors for gene delivery in the early 1980s. However, provirus into the nucleus and thus reduces integration frequency.
since these early studies, a multitude of virus vectors have been Practical issues, including the difficulty in obtaining high-titer virus
developed. All vector systems exploit the virus life cycle to increase in large-scale preparations required for human trials, have also been
the frequency and fidelity of gene transfer. Although many vector noted.
systems have been developed, retrovirus and lentivirus vectors have These difficulties have led to various strategies and the develop-
become the most used platforms for human gene therapy trials ment of entirely new vector systems, which seek to improve gene
involving HSCs, and this review will focus primarily on these vector transfer methods in human HSCs. These strategies include attempts
systems and the closely related foamy virus and avian virus vectors to increase virus–cell interactions or methods to enhance the chances
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(reviewed by Touw and Erkeland ). As noted, the majority of trials of successful DNA integration. Different viral envelopes were used
registered with the Recombinant DNA Advisory Committee of the to pseudotype recombinant particles to more efficiently target CD34
National Institutes of Health use retroviruses, with nonintegrating cells. The use of various cell surface markers, such as CD34, to purify
adenovirus vectors, adeno-associated virus, and nonvirus (liposomes the target cell population can also increase the multiplicity of infec-
and plasmids) systems making up the second- and third-largest tion at a given virus titer and has been used in clinical transplantation
groups. The latter are primarily focused on immune stimulation trials protocols. Thus, the development of antibody-based enrichment of
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in cancer and have limited relevance to the use of HPSCs for the the CD34 HPSC compartment from human hematopoietic tissues
treatment of genetic blood diseases. using magnetic column purification provides a rapid, clinically
applicable method to further enhance retroviral transduction by
increasing the vector-to-target cell ratio. Methods to increase physical
Retrovirus Vectors interactions between vector particles and target cells include colocal-
ization on fibronectin and centrifugation methods. Where polycations
The use of γ-retroviruses as gene transfer vectors takes advantage of such as polybrene had previously been used to enhance transduction
the normal virus life cycle. The virus, a membrane-bound particle frequencies by negating electrostatic charge repulsion between target
enclosing a dimer of genomic RNA, Gag, and reverse-transcriptase cells and viral particles, the characterization of the recombinant
proteins, interacts with specific cell surface receptors on the target CH296 fibronectin fragment (Retronectin) as a matrix upon which
cell. After entry into the cytoplasm, the virus is uncoated, and one could colocalize HSCs and viral particles was a significant
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the genomic messenger (m)RNA is reverse transcribed into DNA. advance in the quest to improve CD34 transduction frequencies.
Subsequent polymerase activity yields a double-stranded (DS) DNA Other efforts to increase the chances of DNA integration have
provirus molecule. For γ-retroviruses, transport into the nucleus focused on attempts to increase the number of HSCs that are under-
depends on the loss of the nuclear membrane, which accompanies going cell division (primarily the use of cytokines that effect stem cell
cell division (see later discussion). Integration of the DS provirus proliferation). The development of improved in vitro growth media
in the chromosome is semi-random. The occurrence of insertional formulations incorporating novel cytokine cocktails achieved the dual
activation of oncogenes in several human trials and the subsequent aim of promoting HSC division, which is required for transduction
scrutiny of insertion sites in HSC-derived progeny in both murine with γ-retroviral vectors while minimizing stem cell loss via apoptosis
and human cells using deep sequencing methods have provided a or differentiation. Finally, the use of new virus systems that do not
more detailed understanding of the subtle but biologically relevant require nuclear membrane disruption (and, therefore, cell division)
preferences for insertions of these vectors (see later discussion). After for entry of the provirus DNA into the nucleus, including primarily
being integrated, the provirus can give rise to mRNA, leading to lentivirus vectors but potentially also vectors based on foamy viruses,
encoded protein products. Full-length (genomic) mRNA can also appears to be the most significant development in the field in the past
be used as the genomic nucleic acid in newly formed virus particles, decade. These newer vector systems will be discussed later.
which are budded nonlytically from the cell surface after assembly In addition to advancing stem cell transduction methodology,
in the cytoplasm of the infected cell. The use of retroviruses for gene additional work has focused on developing retroviral vectors that
delivery depends on the capacity to replace viral genes with other would express transgene cassettes at levels that would be high enough
heterologous gene sequences and to provide necessary viral proteins to elicit a therapeutic benefit and be resistant to gene silencing.
in trans in specialized cell lines, called packaging cells. The advanced Advances in vector design such as the optimization of long-terminal
generation of packaging cells appears to be capable of generating repeat (LTR) enhancer and promoter elements and viral leader
pure stocks of recombinant virus without contaminating wild-type sequences resulted in recombinant vectors that were able to mediate
helper virus, an important safety consideration. Indeed, to date in high-level transgene expression in both primitive and mature hema-
human trials, there have been no reports of inadvertent generation topoietic cells. As discussed in detail later, although these powerful
of infectious virus. Thus, the infection with replication-incompetent promoter and enhancer elements provided robust expression of
(i.e., helper-free) retrovirus vectors would be predicted to yield transgenes, they also appear to be capable of long-range activation of
integration into the targeted cell population but no further spread endogenous regulatory sequences as a form of insertional mutagen-
of virus in the body of the treated patient. The proteins provided in esis, which can have significant deleterious effects. Taken together,
trans for γ-retroviruses are generally Gag, reverse transcriptase, and these technologic advances served as the platform for the first suc-
envelope proteins, the latter defining the host range of infection. cessful gene therapy trial in humans.
In summary, the advantages of retrovirus vectors include the high The use of pharmacologic in vivo selection in combination with
efficiency of stable transfer of intact DNA sequences, the broad gene transfer, both in the setting of cancer trials and in genetic dis-
range of host cells susceptible to infection by retroviruses, and the eases, remains a potentially important method to enhance the
ability to generate helper-free recombinant virus via stable packaging reconstitution of human recipients with gene-modified blood cells,
cell lines. but it has not yet gained widespread usage. General considerations
Despite these advantages, the application of retrovirus vectors for include the need for a particular drug to effect damage to BM stem
treatment of human blood diseases in early trials was disappointing. or progenitor cells. A gene or genes encoding resistance to this agent
In multiple studies, transduction of long-lived and transplantable would need to be identified and resistance in vivo to the agent would
HSCs has been extremely low. In most studies, the frequency of need to be demonstrated after overexpression of this gene in BM cells.
circulating marked blood cells was too low to effect phenotypic For applications in cancer therapies, dose intensification of drugs used
correction of any disease, usually less than 0.1%. The biologic within chemotherapeutic regimens should improve antitumor effi-
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parameters contributing to the poor results in human trials are varied. cacy. Work is ongoing to use transgenic expression of O -methylguanine
The major impediments appear to include the low levels of viral methyltransferase (MGMT), which generates resistance to bis-
receptors on the surface of human HSCs, reducing the efficiency of chloroethylnitrosourea, temazolamide, and 1-(2-chloroethyl)-3-
interaction of virus particles with these target cells, and the quiescent cyclohexyl-1-nitrosourea and cytidine deaminase, which generates
nature of the majority of HSCs, which hinders the transport of the resistance to cytosine arabinoside, gemcitabine, decitobine, and