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Principles of Cell-Based Genetic Therapies
The use of gene transfer to treat human diseases has now been demonstrated to be efficacious in a growing number of indications. Proof-of-principle successes in several monogenic diseases—both hematologic and non-hematologic—have been published and widely publicized in the past decade. The previous occurrence of serious adverse events in some trials related to insertional mutagenesis has stimulated the development of safer vector systems and the improved efficiency of gene editing methods at a clinical scale, particularly those using clustered regularly interspaced short palindromic repeat (CRISPR) technology, holds great promise in the near term. This chapter discusses the basic biology of vector systems applicable to blood diseases, the details of the application of gene therapy to blood diseases using specific trials as examples of this technology, and the modifications in vector systems driven by clinical experience that predict future trials. The chapter also discusses the evolving field of genome editing that is already being tested in clinical trials.
Hematologic Diseases, Cellular Targets, and the Basis for Genetic Therapies
Gene therapy is defined as the introduction of new genetic material into the cells of an organism for therapeutic purposes. Broadly speaking, two types of gene therapy can be envisioned. The introduction of genetic material into germ cells such that the new DNA can be expected to be passed into the gene pool. This is termed germline gene therapy and is currently banned in the United States and around the world. The potential for newer methods of genome engineering to be utilized in clinical applications (discussed later) has led to more recent calls for extending this restriction. In contrast, the introduction of new genetic material into specialized cells of the body with no risk of the new genetic material being passed onto subsequent generations is termed somatic gene therapy . The ultimate goal of gene therapy is to correct the phenotypic abnormalities of the targeted genetic disease by replacement of the defective gene in situ. Such gene replacement could be envisioned via a process termed homologous recombination . Homologous recombination in mammalian cells is widely practiced in laboratories, but up to now, has been relatively inefficient, although newer technology appears to overcome the previous limitations of efficient genome editing. Advantages of this approach would include a reduction in the risk of inadvertent disruption or dysregulation of expression of a critical gene sequence and regulated (appropriate level and distribution) expression of the normal (replaced) gene. However, the frequency of homologous recombination (in contrast to random chromosomal integration or even editing using non-homologous methods) in mammalian cells and primary tissues at a clinical scale makes therapeutic use of homologous recombination still somewhat impractical at this point. Methods to effect homologous recombination efficiency have improved in the past 5 years and may make this goal attainable in the future.
The requirements for successful application of our current gene transfer technology for the treatment of human diseases include knowledge of the abnormal gene sequence responsible for the disease phenotype and the availability of the corresponding normal gene sequence that can be packaged into current vector backbones for efficient recombinant virus production. In addition, the cells responsible 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 be reversed is needed. Although effectively accomplished in the early 1980s in murine studies, this latter requirement encompassing both in vivo administration of DNA sequences and ex vivo cell manipulation has been more difficult to consistently meet in human applications using current gene transfer technology.
Since the early development of virus vectors, blood-forming cells have been used as one optimal target for ex vivo gene transfer studies. For this purpose, hematopoietic stem and progenitor cells (HPSCs) are isolated, manipulated in the laboratory and administered back to the patient. The advantages of these cells as targets of gene transfer are multiple. First, all blood cells are derived from a common progenitor cell, the hematopoietic stem cell (HSC), which is both long-lived in vivo and capable of significant self-renewal. The latter capacity and the pluripotency of HSC is exploited to amplify the genetically manipulated cells into large numbers of progeny cell numbers of multiple blood lineages derived from the genetically altered cells in vivo. There is a long and successful experience in obtaining these stem cells from the bone marrow (BM) and peripheral and umbilical cord blood. There is extensive experience in the use of HSCs in the clinical setting for autologous and allogeneic transplantation, and there is the experience in the purification of these cells and limited knowledge of the requirements for ex vivo manipulation of the cells. In addition, the experience of HSC transplantation (HSCT) has defined a variety of genetic diseases in which the phenotype can be altered by the successful engraftment of normal allogeneic donor cells. Finally, the blood system is involved as a major dose-limiting organ in cancer therapies and both a target and an effector organ in immune reactions provide a large group of diseases that could theoretically be approached using gene transfer technology. As noted earlier, there are already many monogenic diseases of the blood extensively characterized with more being defined at the molecular level on a regular basis as whole-exome and whole-genome sequencing is being applied to rare disease phenotypes. In addition to HSC targets, another application of gene transfer technology exploits the experience in adoptive T-cell immunotherapy. In this application, T cells (and less well-developed to this point, other immune effector cells) are modified ex vivo to enhance potency and specificity. This application of gene transfer technology will not be reviewed here.
The field of gene therapy is rapidly evolving. Successes of “proof-of-principle” small trials have demonstrated the utility of the gene transfer approach in a growing number of patients but in a limited number of diseases leading to the first genetic therapies to be approved by regulatory agencies worldwide as therapeutic medicines. The technology itself is quickly evolving in response to new understandings of viruses, the regulation of gene expression, and improved gene-editing technologies. The application of gene transfer technology to HSC gene therapy has been made possible by the exploitation of viruses that have evolved the capacity to insert viral genomes efficiently and precisely into cellular chromosomes of infected cells. The field has taken >30 years to evolve to its current state of clinical application. Although this might be viewed as a slow pace, this time frame parallels the development of many other novel therapies. This developmental phase also reflects the complexities of the biologic systems involved and the caution required in moving forward in the face of serious adverse events seen in early safety trials. It is indeed an exciting time with respect to the clinical application of gene transfer technology in human diseases.
Vector Systems
The initial impetus to develop gene transfer for human studies derived from the exploitation of oncoretroviruses, mainly murine gamma (γ)-retroviruses, as vectors for gene delivery in the early 1980s. However, since these early studies, a multitude of virus vectors have been developed. All vector systems exploit the virus life cycle to increase the frequency and fidelity of the gene transfer. Although many vector systems have been developed, retrovirus and lentivirus vectors have become the most used platforms for human gene therapy trials involving HSC and T cell targets and this review will focus primarily on these vector systems (reviewed by Touw and Erkeland). As noted, the majority of trials registered with the Recombinant DNA Advisory Committee (RAC) of the National Institutes of Health use retroviruses (both γ-retroviruses and lentiviruses) with non-integrating adenovirus vectors, adeno-associated virus, and nonvirus (liposomes and plasmids) systems making up the second and third largest groups. The latter is primarily focused on immune stimulation trials in cancer and have limited relevance to the use of HPSCs for the treatment of genetic blood diseases.
Retrovirus Vectors
The use of γ-retroviruses as gene transfer vectors takes advantage of the normal virus life cycle. In general, lentivirus vectors exploit a similar lifecycle as a vector system. The virus, a membrane-bound particle enclosing a dimer of genomic RNA, gag, and reverse transcriptase proteins, interacts with specific cell surface receptors on the target cell. After entry into the cytoplasm, the virus is uncoated, and the genomic messenger (m)RNA is reverse transcribed into DNA. Subsequent polymerase activity yields a double-stranded (DS) DNA provirus molecule. For γ-retroviruses, transport into the nucleus depends on the loss of the nuclear membrane, which accompanies cell division (see later discussion). Integration of the DS provirus in the chromosome is semi-random. The occurrence of insertional activation of oncogenes in several human trials and the subsequent scrutiny of insertion sites in HSC-derived progeny in both murine and human cells using deep sequencing methods have provided a more detailed understanding of subtle but biologically relevant preferences for insertions of these vectors (see later discussion). After being integrated, the provirus can give rise to mRNA leading to encoded protein products. Full-length (genomic) mRNA can also be used as the genomic nucleic acid in newly formed virus particles, which are budded nonlytically from the cell surface after assembly in the cytoplasm of the infected cell. The use of retroviruses for gene delivery depends on the capacity to replace viral genes with other heterologous gene sequences and to provide necessary viral proteins in trans in specialized cell lines, called packaging cells . The advanced generation of γ-retroviruses packaging cells appears to be capable of generating pure stocks of the recombinant virus without contaminating wild-type helper virus, an important safety consideration. Indeed, to date in human trials, there have been no reports of inadvertent generation of infectious viruses. Thus, the infection with replication-incompetent (i.e., helper-free) retrovirus vectors would be predicted to yield integration into the targeted cell population but no further spread of the virus in the body of the treated patient. The proteins provided in trans for γ-retroviruses are generally gag, reverse transcriptase, and envelope proteins, the latter defining the host range of infection. In summary, the advantages of retrovirus vectors include the high efficiency of stable transfer of intact DNA sequences, the broad range of host cells susceptible to infection by retroviruses, and for γ-retroviruses the ability to generate helper-free recombinant virus via stable packaging cell lines.
Despite these advantages, the application of retrovirus vectors for the treatment of human blood diseases in early trials was disappointing. In multiple studies, transduction of long-lived and transplantable HSCs was extremely low. In most studies, the frequency of circulating marked blood cells was too low to effect phenotypic correction of any disease, usually less than 0.1%. The biologic parameters contributing to the poor results in human trials are varied. The major impediments appear to include the low levels of viral receptors on the surface of human HSCs, reducing the efficiency of interaction of virus particles with these target cells, and the quiescent nature of the majority of HSC, which hinders the transport of the provirus into the nucleus and thus reduces the integration frequency. Practical issues, including the difficulty in obtaining high-titer viruses in large-scale preparations required for human trials, have also been noted.
These difficulties have led to various strategies and the development of entirely new vector systems, which seek to improve gene transfer methods in human HSCs. These strategies include attempts to increase virus–cell interactions or methods to enhance the chances of successful DNA integration. Different viral envelopes were used to pseudotype recombinant particles to more efficiently target CD34 cells. The use of various cell surface markers, such as CD34, to purify the target cell population can also increase the multiplicity of infection at a given virus titer and has been used in clinical transplantation protocols. Thus, the development of antibody-based enrichment of the CD34 + HPSC compartment from human hematopoietic tissues 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 interactions between vector particles and target cells include co-localization on fibronectin and centrifugation methods. Where polycations such as polybrene had previously been used to enhance transduction frequencies by negating electrostatic charge repulsion between target cells and viral particles, the characterization of the recombinant CH296 fibronectin fragment (Retronectin™) as a matrix upon which one could co-localize HSCs and viral particles was a significant advance in the quest to improve CD34 + transduction frequencies. More recently effective combinations of agents called “transduction enhancers” have been increasingly utilized in clinical trials. Other efforts to increase the chances of DNA integration have focused on attempts to increase the number of HSC that are undergoing cell division (primarily the use of cytokines that affect stem cell proliferation). The development of improved in vitro growth media formulations incorporating novel cytokine cocktails achieved the dual aim of promoting HSC division, which is required for transduction with γ-retroviral vectors and “activation” required for lentivirus vectors while minimizing stem cell loss via apoptosis or differentiation. Finally, the use of new virus systems that do not require nuclear membrane disruption (and, therefore, cell division) for entry of the provirus DNA into the nucleus, including primarily lentivirus vectors, but potentially also vectors based on foamy viruses, appears to be the most significant development in the field in the past decade. Lentivirus vectors will be discussed in more detail later.
In addition to advancing stem cell transduction methodology, additional work has focused on developing retroviral vectors that would express transgene cassettes at levels that would be high enough to elicit a therapeutic benefit and be resistant to gene silencing. Advances in vector design such as the optimization of long-terminal repeat (LTR) enhancer and promoter elements and viral leader sequences resulted in recombinant vectors that were able to mediate high-level transgene expression in both primitive and mature hematopoietic cells. As discussed in detail later, although these powerful promoter and enhancer elements provided a robust expression of transgenes, they appear also to be capable of long-range activation of endogenous regulatory sequences as a form of insertional mutagenesis that can have significant deleterious effects. Taken together, these technological advances served as the platform for the first successful gene therapy trial in humans.
The use of pharmacologic in vivo selection in combination with gene transfer, both in the setting of cancer trials and in genetic diseases, remains a potentially important method to enhance the reconstitution of human recipients with gene-modified blood cells but has not yet gained widespread usage. General considerations include the need for a particular drug to effect damage to BM stem or progenitor cells. A gene’s or genes’ encoding resistance to this agent would need to be identified and resistance in vivo to the agent would need to be demonstrated after overexpression of this gene in BM cells. For applications in cancer therapies, dose intensification of drugs used within chemotherapeutic regimens should improve antitumor efficacy. Work is ongoing to use the transgenic expression of O 6 -methylguanine methyltransferase (MGMT) which generates resistance to bis-chloroethylnitrosourea (BCNU), temazolamide (TMZ) and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) and cytidine deaminase which generates resistance to cytosine arabinoside, gemcitabine, decitobine, and azacytodine for applications in cancer. For non-cancer applications (for the co-selection of a non-selectable therapeutic gene in a genetic disease application), the mutagenic potential of the chemotherapy agent must be considered as a risk in relation to the overall benefit of the gene therapy procedure. Several chemoresistance genes and chemotherapy drug combinations are currently under investigation for this application and encouraging preclinical studies in model systems, including murine and canine studies, have led to a limited number of early-phase human studies in cancer patients.
Lentivirus Vectors
One of the key advances in the gene therapy field in the past decade has been the development of recombinant vectors based on lentiviruses, including the human immunodeficiency virus (HIV). These vectors were originally developed after the observation that targets of HIV included more differentiated cells, such as macrophages, that are often also post-mitotic, suggesting this group of retroviruses has evolved a method of circumventing the block in infection of γ-retroviruses seen in non-dividing cells. Investigators demonstrated that lentivirus vectors derived from HIV were capable of infecting non-dividing neurons after direct injection into the brain. Subsequently, 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 carrying the transgene of interest. To date, most lentivirus vectors use vesicular stomatitis virus as an envelope sequence, which provides a very broad range of target cells susceptible to lentivirus vector transduction. In the case of lentivirus vectors, viral gag and pol, as well as tat and rev proteins, expression are required in trans for efficient virus production along with the envelope proteins. These proteins are usually supplied from separate plasmids, and recombinant lentivirus production is today generally created using the “four plasmid” systems encompassing all the necessary viral proteins on three plasmids and the transfer vector sequences of the fourth plasmid. In addition to gag and pol , the transfer vector contains all the virus regulatory sequences required in cis for packaging an infectious particle, including the psi packaging sequence, integrase, and reverse transcriptase. In almost all clinical applications to date, the recombinant lentivirus vector is generated as a transient supernatant from a population of transfected producer cells rather than a clone and each large-scale batch of viruses has variable yields. Thus, the generation of high-titer recombinant virus is more complicated than γ-retroviruses and remains a significant issue for large-scale, clinical-grade production for use in human trials.
To reduce the chances of the generation of replication-competent retroviruses (RCRs), a major safety concern with HIV, most of the nonessential viral sequences have been removed from currently utilized vectors. This included viv, vpu, vpr , and nef genes and subsequently tat , an important regulator of viral transcription. Recombinant vectors generated without these sequences were demonstrated to efficiently infect a variety of cells. RCR testing is based on sensitive assays to detect a gag protein by p24 immunoassays or polymerase chain reaction. In addition, lentivirus vectors have traditionally been produced using the “self-inactivating design” (SIN) for added safety because of the reduced risk of recombination with and subsequent mobilization of endogenous HIV viruses. The transfer vector thus contains a deletion of the 5′ LTR U3 region and are devoid of viral enhancer and promoter sequences. During reverse transcription, the 5′ LTR is replicated, and the integrated provirus is thus devoid of both 5′ and 3′ enhancers. In SIN constructs, the transgene of interest is thus expressed from an internal promoter that can be chosen with varying strengths or lineage specificities. This added safety feature ultimately has proven important in reducing the risk of insertional mutagenesis (see later) by which integration near cellular genes are inadvertently activated by the LTR enhancer sequences of vectors.
Lentivirus vectors were originally developed for use in a wide range of tissues in which cells are largely non-dividing. Early work focused on the brain, retinal cells, liver, pancreatic islets, airway epithelium, and muscle. However, it was subsequently appreciated that the requirement of stimulating HSCs into division with various cytokines to effect efficient transduction with γ-retrovirus vectors may have negative effects on engraftment or that a large fraction of HSCs remain quiescent and, therefore, resistant to transduction during clinical transduction protocols. After several groups reported successful transduction of primitive HSC populations in protocols in which these cells remained resistant to transduction by γ-retroviruses, the adoption of lentivirus vectors has now included multiple human trials with encouraging results. One of the earliest trials to utilize lentivirus vectors was in childhood cerebral adrenoleukodystrophy (CCALD) in which long-term “marking” in the myeloid compartment appears to be 10% to 20%, a level that is 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 design used in all lentivirus vectors, there is an additional theoretical advantage of the preference of lentiviruses for integration away from transcription start sites (TSS) of genes in contrast to γ-retroviruses. However, the recent experience with a lentivirus vector used in a single patient with thalassemia in which abnormal splicing resulted in clonal expansion in the erythroid compartment, suggests that insertion within genes including intronic and non-coding regions of genes may also have potential adverse effects on endogenous sequences. These trials are described in more detail later. Thus, the long-term safety of lentiviruses in human trials remains to be determined, and important aspects of insertional mutagenesis are described in more detail later.
Experience in Hematologic Clinical Trials to Date
X-Linked Severe Combined Immunodeficiency
Severe combined immunodeficiency (SCID) comprises a number of rare monogenic diseases with the common feature of a block in T-cell differentiation and impaired B-cell and natural killer (NK) cell immunity. Studies of the pattern of inheritance, immune function, and genotypes have led to the identification of at least 18 distinct SCID conditions with the number increasing as genomic sequencing identifies mutations in additional immunodeficiency phenotypes. The most common variant of SCID results from the deficiency in the expression or function of the common cytokine receptor γ chain, which is shared by the receptors for interleukin (IL)-2, IL-4, IL-7, IL-9, IL-15, and IL-21. This condition is inherited in a sex-linked fashion (X-linked SCID or SCID-X1) and accounts for ~30% of all SCID cases. SCID-X1 is characterized by abnormal development or function of T, B, and NK cells, although B cells are usually present in humans (so-called T-minus, B-plus SCID). Survival depends on the reconstitution of T-cell development and function by allogeneic BM transplantation (BMT). If a genotypically matched family donor is available, HSCT confers a greater than 80% chance of long-term survival. The absence of T and NK cells in the patient allows for the engraftment of donor cells without preparative chemotherapy conditioning; thus, this is the treatment of choice with minimal toxicity. When a genotypically matched family member is not available, haploidentical donors (e.g., a parent) or closely matched unrelated donors are used, with varying preference from center to center, and a survival rate of 64% to 78% has been reported. These inferior outcomes may be attributed to the increased risk of graft rejection or graft-versus-host disease (GVHD), as well as the effects of T-cell depletion, immune suppression causing slower immune reconstitution, or conditioning with an increased risk of infection. Haploidentical transplants rigorously depleted of T cells, similar to genotypically related transplants, are performed in some institutions without preparative chemotherapy conditioning; however, B-cell reconstitution is poor, and the majority of patients require intravenous immunoglobulin (IVIG) replacement for life. Interestingly, spontaneous partial correction of severe T-cell immunodeficiencies, including SCID, has previously been reported, suggesting a selective advantage of wild-type T cells over defective T cells.
Multiple independent gene therapy trials aimed at correcting the immunologic defect of SCID-X1 patients who lack a genotypically matched BM donor have been reported. In the initially reported trials, done in Paris and London, a total of 20 patients have been treated. Despite minor technical differences in the two protocols, the basic design of both gene therapy trials is quite similar: The complete coding region of the human γ chain was cloned into a “first-generation” γ-retroviral vector regulated by the murine leukemia virus (MLV) LTR sequences, which was used to infect BM-derived CD34 + cells in vitro. Results in both trials have been extremely encouraging.
In the French trial, 10 children younger than the age of 1 year were enrolled between 1999 and 2002. Nine of 10 infants developed normal numbers of T and NK cells, with good immune function. In seven of the nine patients who developed T cells, T-cell counts reached normal levels within three months and have remained normal at the time of the last published follow-up. Protective levels of antibodies, including antibody production after immunization, were achieved, and the prophylactic administration of IVIG was discontinued. At almost >10 years after gene therapy, these patients continued to retain a functional immune system, enabling them to live normally.
However, serious adverse events related to gene therapy have been reported in five patients in the French trial, occurring up to 15 years after gene therapy and in one patient in the British trial. In these patients, untoward effects of viral integration into the genome resulted in T-cell leukemia, leading to the death of one of the affected patients. Much research has subsequently been directed at elucidating the mechanism responsible for these adverse events. Retroviral integration in the proximity of proto-oncogenes, particularly the LIM domain only 2 ( LMO2 ) promoter, was involved in leukemogenesis in three French patients and one British patient. Integration of the unaltered γ chain–encoding viral vector on chromosome 11q13, near the first exon of the LMO2 gene, led to the unregulated transcription of LMO2, giving rise to a T-cell acute lymphoblastic leukemia (T-ALL)–like lymphoproliferation in the initial two patients. In the patient in the British trial, the integration of the vector 35 kb upstream of the LMO2 locus cooperated with secondary genetic aberrations, including a gain of function mutation of NOTCH1, a deletion at the CDKN2A tumor suppressor gene locus, and translocation of the T-cell receptor β region, to give rise to T-ALL. LMO2 is a master regulator of human hematopoiesis that is involved in stem cell growth and is not normally expressed in T cells. However, LMO2 activation has been implicated in some cases of human T-cell leukemia. In addition, LMO2 transgenic mice have been shown to develop T-ALL within 10 months. It is increasingly clear that retroviral vectors may “turn on” cellular proto-oncogenes adjacent to their integration site in the genome. The strong promoter or enhancer activity of the retroviral LTR element shows a propensity to the upregulation of genes neighboring the integration site. Multiple studies now indicate that γ-retroviral vectors, such as the vectors used in the two SCID-X1 trials, preferentially integrate into the 5′ end of genes near the TSS. In addition, γ-retroviral vectors have been shown to integrate into or near a number of proto-oncogenes that are actively expressed in human CD34 + cells. When human CD34 + cells were transduced with retroviral vectors ex vivo, 21% of retroviral integrations occurred at recurrent insertion sites (“hot spots”), which were highly enriched for proto-oncogenes and growth-controlling genes. A series of papers investigating the vector integration sites in both SCID-X1 trials and a trial treating Adenosine Deaminase (ADA) Deficiency SCID trial (see below) observed a greater-than-random frequency of vector integrations near the TSS of genes that are active in HSCs. Interestingly, in the SCID-X1 trial, a skewing of vector integration site distribution in vivo was noted. Compared with retroviral integration sites (RIS) recovered from transduced CD34 + cells, RIS recovered from T cells in vivo 9 to 30 months after transplantation showed an overrepresentation of RIS within or near genes encoding proteins with kinase activity, transferase activity, or proteins involved in phosphorous metabolism. This skewing of RIS in vivo suggests a selection of T cells as a result of viral integration in certain growth- and survival-promoting genes. A subsequent trial in X-SCID utilizing a SIN γ-retrovirus design (NCT01129544) reports follow-up on nine patients, although the trial has now closed for accruing patients. The virus design is noteworthy in that it is more directly comparable to a lentivirus since the viral enhancer elements are deleted and the expression of the IL-2 γ chain is from an “internally positioned” cellular promoter. The initial results reported efficacy similar to the previous SCID-X1 trials, no leukemia and integration analysis that appears safer than those seen in the previous two trials. In the eight evaluable patients treated in this trial, six demonstrated correction of T-cell reconstitution. One patient died prior to full engraftment of gene-modified cells of pre-existing viral infection that did not resolve after gene therapy. The two failures correlated with lower VCN in the transduced product as a result of inadequate gene transfer. This study is particularly noteworthy as the only human study in which a γ-retrovirus vector deleted of enhancer elements has been utilized. It thus provides a platform for more direct comparison with previous γ-retrovirus and lentivirus trials with respect to safety and efficacy. More recently, a trial using similar cis-regulatory elements driving the gamma chain cDNA in a lentivirus vector has been reported, which includes the successful reconstitution of the B-cell compartment after including conditioning with busulfan.
In summary, thus far, six of 20 patients treated with gene therapy for SCID-X1 have encountered a life-threatening severe adverse event, thought to be triggered by retroviral activation of LMO2 in four patients. Four patients were salvaged with chemotherapy, and one patient succumbed to the disease after an unsuccessful allogeneic BMT. At this point, the use of MLV-based retroviral vectors with LTR promoter enhancer elements intact is viewed as contraindicated in this disease by most investigators in the field. The continued development of safety-enhanced vectors and the validation of these vectors in clinically relevant systems have emerged as a major priority in the field, and an international trial has recently been reported using a γ-retrovirus that is deleted of LTR enhancer elements. Transgene expression is mediated by a weak cellular promoter in this vector. Data suggest that early efficacy is maintained despite lower expression of the cγ chain and that the deletion of the γ-retrovirus enhancer is a key to improved safety.
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