Page 1746 - Hematology_ Basic Principles and Practice ( PDFDrive )

Chapter 98 Principles of Cell-Based Genetic Therapies 1551

azacytodine for applications in cancer. For noncancer applications epithelium, and muscle. However, it was subsequently appreciated
(for coselection of a nonselectable therapeutic gene in a genetic that the requirement of stimulating HSCs into division with various
disease application), the mutagenic potential of the chemotherapy cytokines to effect efficient transduction with γ-retrovirus vectors may
agent must be considered as a risk in relation to the overall benefit have negative effects on engraftment, or that a large fraction of HSCs
of the gene therapy procedure. Several chemoresistance genes and remain quiescent and therefore resistant to transduction during clini-
chemotherapy drug combinations are currently under investigation cal transduction protocols. After several groups reported successful
for this application, and encouraging preclinical studies have led to transduction of primitive HSC populations in protocols in which
a limited number of early-phase human studies in cancer patients. In these cells remained resistant to transduction by γ-retroviruses, the
a recently reported human trial in which MGMT gene transfer was adaption of lentivirus vectors has now included several recent human
utilized, patients receiving temazolamide for treatment of brain trials, and initial results are encouraging. In a trial for childhood
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tumors tolerated significantly more chemotherapy compared with cerebral adrenoleukodystrophy (CCALD), long-term “marking” in
historical controls, with transient increases in gene-modified cells in the myeloid compartment appears to be 10% to 20%, a level that is
the peripheral blood in a few patients. 2 about 100-fold higher than the marking in the myeloid compartment
seen in previous trials in immunodeficiency conditions that used
γ-retrovirus vectors. In addition to the safety advantage of SIN vector
Lentivirus Vectors design used in all lentivirus vectors, there is an additional theoretical
advantage of the preference of lentiviruses for integration away from
One of the key advances in the gene therapy field in the past decade transcription start sites (TSS) of genes in contrast to γ-retroviruses.
has been the development of recombinant vectors based on lentivi- However, the recent experience with a lentivirus vector used in a
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ruses, including human immunodeficiency virus (HIV). These single patient with thalassemia, in which abnormal splicing resulted
vectors were originally developed after the observation that targets of in clonal expansion in the erythroid compartment, suggests that
HIV included more differentiated cells, such as macrophages, that insertion within genes including intronic and noncoding regions of
are often also postmitotic, suggesting this group of retroviruses has genes may also have potential adverse effects on endogenous sequences.
evolved a method of circumventing the block in infection of These trials are described in more detail later. Thus, the long-term
γ-retroviruses seen in nondividing cells. Investigators demonstrated safety of lentiviruses in human trials remains to be determined, and
lentivirus vectors derived from HIV were capable of infecting non- important aspects of insertional mutagenesis are described in more
dividing neurons after direct injection into the brain. Subsequently, detail later.
Naldini and colleagues showed efficient infection of growth-arrested
cells. As with γ-retroviruses, lentivirus vectors use key viral gene
products in trans to generate replication-defective infectious particles Foamy Virus Vectors
carrying the transgene of interest. To date, most lentivirus vectors use
vesicular stomatitis virus as envelope sequence, which provides a very Foamy viruses possess several features that have been exploited to
broad range of target cells susceptible to lentivirus vector transduc- yield vectors for the purpose of transducing HSCs. Foamy viruses are
tion. In the case of lentivirus vectors, viral Gag and Pol, as well as members of the spumaretroviruses family. They have been shown to
Tat and Rev protein expression are required in trans for efficient virus be endemic retroviruses in a wide range of animals but are not found
production along with the envelope proteins. These proteins are in humans. These vectors are the largest of the retroviruses (≈14 kb)
usually supplied from separate plasmids, and recombinant lentivirus and thus yield vectors with a capacity to efficiently package large
production is today generally created using “four-plasmid” systems amounts of genetic sequences. The virus has a DNA genome that is
encompassing all the necessary viral proteins on three plasmids and reversed transcribed within the virion particle, forming a stable pre-
the transfer vector sequences on the fourth plasmid. In addition to integration complex within the transduced cell. As with γ-retrovirus
gag and pol, the transfer vector contains all the virus regulatory vectors, the virus packaging signals have been successfully exploited
sequences required in cis for packaging an infectious particle, includ- to allow production of high-titer, replication-free vector stocks. Based
ing the psi packaging sequence, integrase, and reverse transcriptase. on accidental exposure of a limited number of animal care workers,
Thus, generation of high-titer recombinant virus is more complicated it has been reported that infection of humans with wild-type foamy
than γ-retroviruses and remains a significant issue for large-scale, virus has no pathologic effects. Foamy virus vectors have been used
clinical grade production for use in human trials. to successfully transduce mouse and human hematopoietic cells.
To reduce the chances of generation of replication-competent Proof-of-principle work by Hickstein’s group has demonstrated cor-
retroviruses (RCRs), a major safety concern with HIV, most of the rection of canine leukocyte adhesion deficiency using a foamy virus
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nonessential viral sequences have been removed from currently uti- vector expressing CD18 after transduction and infusion of CD34
lized vectors. This included viv, vpu, vpr, and nef genes, and subse- cells after submyeloablative conditioning. No human trials have been
quently tat, an important regulator of viral transcription. Recombinant opened to date using this vector system, but several are planned for
vectors generated without these sequences were demonstrated to the future.
efficiently infect a variety of cells. RCR testing is based on sensitive
assays to detect Gag protein by p24 immunoassays or polymerase
chain reaction. In addition, lentivirus vectors have traditionally been Alpharetroviruses
produced using the “self-inactivating design” (SIN) for added safety
because of reduced risk of recombination with and subsequent Retrovirus vectors derived from Rous sarcoma virus, which is a
mobilization of endogenous HIV viruses. The transfer vector thus member of the alpharetrovirus family, have been described. Although
contains a deletion of the 5′ LTR U3 region and is devoid of viral these vectors maintain genomic integration as part of the viral life
enhancer and promoter sequences. During reverse transcription, the cycle, they have attracted recent attention because of their propensity
5′ LTR is replicated, and the integrated provirus is thus devoid of to integrate in a relatively neutral fashion with respect to promoter
both 5′ and 3′ U3 regions. In SIN constructs, the transgene of interest regions and TSS of genes. In large animal studies, alpharetrovirus-
is thus expressed from an internal promoter that can be chosen with transduced HSC-derived progeny demonstrated integrations that
varying strengths. This added safety feature ultimately has also proven were not clustered in gene-rich CpG or TSS regions of the genome.
important to reduce the risk of insertional mutagenesis (see later), by The development of a self-inactivating alpharetrovirus with a split-
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which integration near cellular genes are inadvertently activated by packaging design by Schambach and colleagues has provided proof-
the LTR enhancer sequences of vectors. of-principle with respect to the capacity to generate high-titer,
Lentivirus vectors were originally developed for use in a wide replication-free vector stocks from stable producer cell lines that use
range of tissues in which cells are largely nondividing. Early internal promoters to express transgenes in a potentially safer fashion
work focused on brain, retinal cells, liver, pancreatic islets, airway than γ-retrovirus vectors. Such vectors have been demonstrated to