Human Gene Therapy Trials Research Articles

Reduced Genotoxicity of Avian Sarcoma Leukosis Virus Vectors in Rhesus Long-term Repopulating Cells Compared to Standard Murine Retrovirus Vectors

Insertional mutagenesis continues to be a major concern in hematopoietic stem-cell gene therapy. Nonconventional gene transfer vectors with more favorable integration features in comparison with conventional retrovirus and lentivirus vectors are being developed and optimized. In this study, we report for the first time a systematic analysis of 198 avian sarcoma leukosis virus (ASLV) insertion sites identified in rhesus long-term repopulating cells, and a comparison of ASLV insertion patterns to Moloney murine leukemia virus (MLV) (n = 396) and simian immunodeficiency virus (SIV) (n = 289) using the newly released rhesus genome databank. Despite a weak preference toward gene-coding regions, ASLV integration is nonclustered, does not favor gene-rich regions, transcription start sites, or CpG islands. There was no propensity for ASLV insertions within or near proto-oncogenes, and most importantly, no insertions close to or within the Mds1-Evi1 locus, which is in contrast to the significant over-representation of this insertion site for MLV vectors in the same transplantation model. Furthermore, ASLV long terminal repeats (LTRs) do not have detectable promoter and enhancer activity in a quantitative luciferase assay to measure neighboring gene activation. The combination of these features is unique for ASLV and suggests that optimized vectors based on this virus could be useful and safe for gene transfer to hematopoietic stem cells and progenitor cells.

Leber Congenital Amaurosis: From Darkness to Spotlight

Almost 150 years ago, Theodor Leber described a severe form of vision loss at or near birth which was later given his name. During the century that followed this description, ophthalmologists dedicated efforts to give an accurate definition of the disease but patients were neglected because of the inability of physicians to provide them with treatment. In the 90s, at the time of the Golden Age of Linkage, the first LCA locus was mapped to a human chromosome and shortly after identified as the gene for guanylate cyclase. This discovery was the spark that made the disease emerge from the shadows as illustrated by the flood of LCA genes identified in the following ten-year period. During the same time period, the clinical variability of the disease was rediscovered and an unexpected physiopathological heterogeneity demonstrated. In the beginning of the third millennium, LCA came out definitively from the tunnel to shine under the bright spotlights with the RPE65 gene therapy trial that succeeded to restore vision in a dog model and opened the door to gene therapy trials in humans.

In Vivo Biosafety Model To Assess Risk of Adverse Events from Retroviral and Lentiviral Vectors.

Due to a heightened awareness of the potential for adverse events during human gene therapy trials, it is important to document the safety of the vectors and to examine the potential for malignancy arising from insertional mutagenesis from integrating vectors, using detailed in vivo analyses. In the current studies, we assessed the potential for generation of replication-competent retrovirus (RCR) and insertional mutagenesis causing adverse events, in human cells transplanted into immune deficient mice. Both retroviral and lentiviral vectors were assessed, in a total of 630 recipient mice. Human hematopoietic and mesenchymal stem cells carrying two different Moloney-based vectors were co-transplanted into beige/nude/XID and NOD/SCID mice that are unable to reject cells that become transformed. A total of 481 mice transplanted with human cells transduced by Moloney-based vectors, and an additional 149 mice that had received human cells transduced by HIV-based lentiviral vectors were monitored daily for adverse events for 2–18 months post-transplantation. An “adverse event” was logged in the experiment, the mouse was immediately sacrificed, and an autopsy was performed if ANY signs of ill health were observed, including any of the following: weight loss, hunching, lethargy, rapid breathing, skin discoloration or irregularities, bloating, hemi-paresis, enlarged lymph nodes or visible solid tumors under the skin. The presence of tumors and organ or lymph node abnormalities, or progression to monoclonal expansion in hematopoietic cells were specifically sought. The organs, marrow, blood, and serum were banked. Tissue sections were prepared from all organs (and visible tumor, if present). DNA was isolated from marrow, organs, blood, and any tumors to determine whether vector elements were present. Of the 630 recipient mice, 117 had some evidence of adverse effects ranging from development of leukemia to scarred skin or discolored organs detected upon autopsy. Human leukemic cells were found infiltrating the murine liver, spleen, blood and lymph nodes in 4 of these 117 mice. No vector DNA was present. 37 of the mice had developed solid tumors, determined to be of murine origin with no vector DNA present or RCR detected in their serum or tumor tissue. Despite the injection of 459 million transduced cells throughout the experiments, the engineered human MSC never gave rise to solid tumors. None of the serum samples or affected tissues from these mice had vector integrants or produced RCR in the marker rescue assay. In summary, 4 of the 117 total adverse events were due to the spontaneous development of human leukemia and the remainder of the events were due to development of cancerous states in the murine tissues, with no evidence of retroviral vector involvement. There was no evidence of insertional mutagenesis causing human leukemia or solid tumors in any mouse. No RCR were detected in 117 serum samples analyzed by vector rescue assay. No adverse events were caused by the vectors and no mouse tested had HIV p24 capsid protein in their serum. The current studies provide an in vivo system to assess potential risk from RCR and from insertional mutagenesis when retroviral or lentiviral vectors are considered as candidates for human gene therapy.

Inhibition of HIV-1 replication with designed miRNAs expressed from RNA polymerase II promoters

Small interfering RNA (siRNA) mediates sequence-specific RNA cleavage and represents a potential approach to treat the infection of human immunodeficiency virus (HIV). Expression of a single siRNA species frequently led to the emergence of HIV escape variants. Thus, multiple siRNAs targeted to different regions in the HIV-1 genome may be required. However, overexpression of different anti-HIV siRNA genes from multiple pol III promoters can induce cell toxicity, thus may not be a viable option in the setting of human gene therapy trials. In the current study, we evaluated the strategy of using pol II promoters to drive the expression of siRNAs against HIV-1. We replaced the stem sequence in the stem-loop structure of the well-characterized miR-30a with siRNA sequences and showed that designed microRNA (miRNA) could be expressed from pol II promoters. We demonstrated efficient inhibition of HIV-1 replication with such designed miRNA, but the efficacy was directly correlated with the expression level. Both the vector copy number and the promoter strength directly affected the ability of the siRNA to inhibit HIV-1 replication. We also showed that a combination of pol II and pol III promoters to express two different siRNAs increased the efficacy against HIV-1 replication without comprising cell viability.

Retroviral integration and human gene therapy

Long-term correction of genetic diseases requires permanent integration of therapeutic genes into chromosomes of affected cells. Retroviral vectors are the most widely used delivery vehicles because of their efficiency and precision of integration. However, retroviral integration can take place at a variety of chromosomal sites, and examples have been reported of integration of therapeutic vectors activating oncogenes and causing cancer in patients. This issue of the JCI presents three articles that update successful human gene therapy trials and furthermore evaluate the sites of integration in cells from treated patients, including samples from individuals experiencing serious adverse events following therapy (see the related articles beginning on pages 2225, 2233, and 2241).

Cytokine-independent growth and clonal expansion of a primary human CD8+ T-cell clone following retroviral transduction with the IL-15 gene

AbstractMalignancies arising from retrovirally transduced hematopoietic stem cells have been reported in animal models and human gene therapy trials. Whether mature lymphocytes are susceptible to insertional mutagenesis is unknown. We have characterized a primary human CD8+ T-cell clone, which exhibited logarithmic ex vivo growth in the absence of exogenous cytokine support for more than 1 year after transduction with a murine leukemia virus–based vector encoding the T-cell growth factor IL-15. Phenotypically, the clone was CD28−, CD45RA−, CD45RO+, and CD62L−, a profile consistent with effector memory T lymphocytes. After gene transfer with tumor-antigen–specific T-cell receptors, the clone secreted IFN-γ upon encountering tumor targets, providing further evidence that they derived from mature lymphocytes. Gene-expression analyses revealed no evidence of insertional activation of genes flanking the retroviral insertion sites. The clone exhibited constitutive telomerase activity, and the presence of autocrine loop was suggested by impaired cell proliferation following knockdown of IL-15Rα expression. The generation of this cell line suggests that nonphysiologic expression of IL-15 can result in the long-term in vitro growth of mature human T lymphocytes. The cytokine-independent growth of this line was a rare event that has not been observed in other IL-15 vector transduction experiments or with any other integrating vector system. It does not appear that the retroviral vector integration sites played a role in the continuous growth of this cell clone, but this remains under investigation.

A Model for Optimizing Adenoviral Delivery in Human Cancer Gene Therapy Trials

Optimization of adenoviral delivery to the target volume is required for adenovirus-mediated cancer gene therapy to reach its maximal potential. The purpose of these studies was to develop a model of gene expression to improve adenovirus-mediated cancer gene therapy in the clinic. We measured the distribution of gene expression after a single deposit of a replication-competent adenovirus carrying the human sodium iodide symporter (hNIS) reporter gene was delivered to naive canine prostate and to human tumor xenografts. We generated hypothetical treatment plans for two prospective prostate cancer patients, using standard brachytherapy algorithms. In both models, the gene expression distribution from a single adenoviral deposit could be accurately described by a Gaussian function. In the naive canine prostate, a 0.1-ml deposit of 3 x 10(11) viral particles (VP) resulted in a gene expression volume of 1.14 +/- 0.70 cm(3), indicating that a minimum of 40 adenoviral deposits would be required to cover a 40-cm(3) prostate with therapeutic gene expression. On a viral particle basis, the gene expression volume obtained in human tumor xenografts (7 x 10(-12) cm(3)/VP) was twice that (3.5 x 10(-12) cm(3)/VP) measured in the naive canine prostate. Hypothetical treatment plans for two prostates indicated that 26 and 57 0.1-ml adenoviral deposits would be required to cover, respectively, 24- and 49-cm(3) prostates with gene expression. Although our studies focused on prostate, we believe the methodology to model gene expression presented here has much broader application to optimize treatment plans in other solid tumor sites; this assertion should be confirmed experimentally.

Gene transfer therapy for neurodegenerative disorders

Recent advances in gene transfer technology have led to promising new therapies for neurodegenerative disorders. This article will review methods of gene transfer therapy and applications of these techniques to both genetic and sporadic neurodegenerative illnesses. The article will focus on Parkinson's disease, Huntington's disease, and Alzheimer's disease. Several promising gene therapy approaches to these diseases are being pursued both in animal models and in early human trials. Initial safety-tolerability results from these trials appear promising. It is therefore likely that the number of human trials of gene therapy for neurodegenerative disorders will increase over the coming years.

Mechanism of Ad5 Vaccine Immunity and Toxicity: Fiber Shaft Targeting of Dendritic Cells

Recombinant adenoviral (rAd) vectors elicit potent cellular and humoral immune responses and show promise as vaccines for HIV-1, Ebola virus, tuberculosis, malaria, and other infections. These vectors are now widely used and have been generally well tolerated in vaccine and gene therapy clinical trials, with many thousands of people exposed. At the same time, dose-limiting adverse responses have been observed, including transient low-grade fevers and a prior human gene therapy fatality, after systemic high-dose recombinant adenovirus serotype 5 (rAd5) vector administration in a human gene therapy trial. The mechanism responsible for these effects is poorly understood. Here, we define the mechanism by which Ad5 targets immune cells that stimulate adaptive immunity. rAd5 tropism for dendritic cells (DCs) was independent of the coxsackievirus and adenovirus receptor (CAR), its primary receptor or the secondary integrin RGD receptor, and was mediated instead by a heparin-sensitive receptor recognized by a distinct segment of the Ad5 fiber, the shaft. rAd vectors with CAR and RGD mutations did not infect a variety of epithelial and fibroblast cell types but retained their ability to transfect several DC types and stimulated adaptive immune responses in mice. Notably, the pyrogenic response to the administration of rAd5 also localized to the shaft region, suggesting that this interaction elicits both protective immunity and vector-induced fevers. The ability of replication-defective rAd5 viruses to elicit potent immune responses is mediated by a heparin-sensitive receptor that interacts with the Ad5 fiber shaft. Mutant CAR and RGD rAd vectors target several DC and mononuclear subsets and induce both adaptive immunity and toxicity. Understanding of these interactions facilitates the development of vectors that target DCs through alternative receptors that can improve safety while retaining the immunogenicity of rAd vaccines.

Ex Vivo Expansion of Retrovirally-Transduced Primate CD34+ Cells Results in Preferential Engraftment and Persistence of Clones with MDS1/EVI1 Insertion Sites.

There is increasing evidence that insertional activation of proto-oncogenes by retroviral vectors is a significant safety issue that must be addressed before clinical gene therapy, particularly targeting hematopoietic stem and progenitor cells, can be further developed. The risk of insertional mutagenesis for replication-incompetent retroviral vectors has been assumed to be low until the occurence of T cell leukemias in children treated with HSC-directed gene therapy for X-SCID, and recent evidence that retroviral integration is more common in the promoter region of transcriptionally-active genes. The occurence of “common integration sites” in a particular gene also suggests a non-random insertion pattern, and/or immortalization or other change in the behavior of a clone harboring an insertion in these particular genes. We have previously reported a highly non-random occurence of 14 unique vector integrations in the first two introns of the MDS1/EVI1 proto-oncogene out of a total of 702 identified from myeloid cells of 9 rhesus macaques at least 6 months post-transplantion of retrovirally-transduced CD34+ cells.(Calmels et al, 2005). This same gene locus was found frequently activated by insertions in murine bone marrow cells immortalized in long-term in vitro culture after transduction with retroviral vectors.(Du et al Blood, 2005) To begin to investigate the factors contributing to this worrisome finding, particularly given the very recent report of a marked over-representation of MDS1/EVI1 insertions in a human clinical gene therapy trial for chronic granulomatous disease, we asked whether continued ex vivo expansion of transduced CD34+ cells prior to transplantation would further select for clones with insertions in MDS1/EVI1 or other proto-oncogenes. Rhesus CD34+ cells were transduced with the G1Na standard retroviral vector, identical to that used in the prior studies, using our standard 96 hour transduction protocol in the presence of Retronectin and SCF, FLT3L and thrombopoietin. At the end of transduction, all cells were continued in culture for an additional 7 days under the same culture conditions, and then reinfused into the donor animal following 1200 rads TBI. At 1 month post-transplant there were no CIS and no MDS1/EVI1 insertions identified. However, at 6 months post-transplantation 5 out of 27 (19%) of the unique insertions identified in granulocytes were within the first two introns of MDS1/EVI1, very significantly higher than the 2% of MDS1/EVI1 insertions (14 of 702) identified in animals that were transplanted with cells not subjected to additional ex vivo expansion.(p<.0001) One MDS1/EVI1 clone constituted 14% of overall sequences identified, and the 5 clones constituted 37% of total sequences identified. This strongly suggests that the over-representation of this locus in engrafting cells is due to a potent immortalizing signal provided by activation of the MDS1/EVI1 gene products by the stonger retroviral promoter/enhancer, and that the need for extended ex vivo culture of target cells may select for insertion events activating this locus. It also suggests that strategies involving prolonged ex vivo expansion or selection of transduced cells could increase the risk of gene therapy utilizing integrating vectors targeting primitive hematopoietic cells.

Skin gene therapy for acquired and inherited disorders.

The rapid advances associated with the Human Genome Project combined with the development of proteomics technology set the bases to face the challenge of human gene therapy. Different strategies must be evaluated based on the genetic defect to be corrected. Therefore, the re-expression of the normal counterpart should be sufficient to reverse phenotype in single-gene inherited disorders. A growing number of candidate diseases are being evaluated since the ADA deficiency was selected for the first approved human gene therapy trial (Blaese et al., 1995). To cite some of them: sickle cell anemia, hemophilia, inherited immune deficiencies, hyper-cholesterolemia and cystic fibrosis. The approach does not seem to be so straightforward when a polygenic disorder is going to be treated. Many human traits like diabetes, hypertension, inflammatory diseases and cancer, appear to be due to the combined action of several genes and environment. For instance, several wizard gene therapy strategies have recently been proposed for cancer treatment, including the stimulation of the immune system of the patient (Xue et al., 2005), the targeting of particular signalling pathways to selectively kill cancer cells (Westphal and Melchner, 2002) and the modulation of the interactions with the stroma and the vasculature (Liotta, 2001; Liotta and Kohn, 2001).

2278: Design Considerations for Incorporating Reporter Gene Imaging Into Human Cancer Gene Therapy Trials

2278: Design Considerations for Incorporating Reporter Gene Imaging Into Human Cancer Gene Therapy Trials

Heparin binding directs activation of T cells against adeno-associated virus serotype 2 capsid

Activation of T cells to the capsid of adeno-associated virus (AAV) serotype 2 vectors has been implicated in liver toxicity in a recent human gene therapy trial of hemophilia B. To further investigate this kind of toxicity, we evaluated T-cell responses to AAV capsids after intramuscular injection of vectors into mice and nonhuman primates. High levels of T cells specific to capsids of vectors based on AAV2 and a phylogenetically related AAV variant were detected. Vectors from other AAV clades such as AAV8 (ref. 3), however, did not lead to activation of capsid-specific T cells. Through the generation of AAV2-AAV8 hybrids and the creation of site-directed mutations, we mapped the domain that directs the activation of T cells to the RXXR motif on VP3, which was previously shown to confer binding of the virion to heparan sulfate proteoglycan (HSPG). Evaluation of natural and engineered AAV variants showed direct correlations between heparin binding, uptake into human dendritic cells (DCs) and activation of capsid-specific T cells. The role of heparin binding in the activation of CD8(+) T cells may be useful in modulating the immunogenicity of antigens and improving the safety profile of existing AAV vectors for gene therapy.

Screening of Novel Excipients for Improving the Stability of Retroviral and Adenoviral Vectors

In the past decade there has been an increase in the application of viral vectors in the laboratory and clinical trials of human gene therapy, retroviral and adenoviral vectors among the most used. However, the limited stability of these vectors creates problems in the design of experiments, transport, and storage. Vectors stored at -80 degrees C must be quickly shipped on dry ice, which is somewhat cumbersome. Alternatively, viral vectors can be preserved in a lyophilized form. However, loss of viral activity during lyophilization can also be a serious problem. In this report we identify novel candidate formulations containing new compatible solutes, ectoin, hydroxyectoin, and firoin, that allow better stability of retroviral and adenoviral vectors during storage. For retroviral vectors, the maximum stabilization for long-term storage was achieved through lyophilization followed by storage at -20 degrees C using a formulation of Tris buffer pH 7.2 containing firoin (0.5 M), a half-life of 340 days being obtained. Adenoviral vectors storage at -80 degrees C in solution using Tris buffer pH 8.0 with firoin was the best method for long-term storage, with a half-life exceeding 1 year.

Gene therapy for immune disorders: Good news tempered by bad news

After a dozen years of human gene therapy trials characterized by minimal gene correction and disappointing clinical impact, the field of gene therapy received some good news in 2000. Infants with X-linked severe combined immunodeficiency who received retroviral gene addition to cells from their bone marrow developed impressive immune reconstitution. During the following 2 years, additional patients were treated and the news was even better-babies receiving gene therapy had sustained T-cell production and in several cases developed better cell function than most patients treated with standard bone marrow transplants. Unfortunately, bad news followed. Three of the patients experienced leukemic T-cell expansions, found to be associated with retroviral insertions into genomic DNA. Where does the field stand today?

662. Retroviral Insertion Site Analysis Following Therapeutic Gene Transfer of CD18 in a Canine Model of Leukocyte Adhesion Deficiency

Retroviral insertion site analysis following the infusion of gene-corrected hematopoietic stem cells has become increasingly important following the development of leukemia in three children with common |[gamma]|-chain severe combined immunodeficiency treated with retroviral gene therapy. This analysis is of particular importance in human gene therapy trials and in gene-transfer studies in disease- specific, large-animal models. We have conducted pre-clinical gene therapy in canine leukocyte adhesion deficiency (CLAD), the canine equivalent of leukocyte adhesion deficiency, type 1 (LAD-1) in humans. Both diseases are characterized by life-threatening bacterial infections and are due to mutations in the leukocyte integrin CD18. We have demonstrated reversal of the disease phenotype in six dogs with CLAD following infusion of autologous CD34+ bone marrow hematopoietic stem cells transduced with the retroviral vector PG13/MSCV-cCD18 after non-myeloablative conditioning with either 200 cGy TBI or 10 mg/kg busulfan. In the six CLAD dogs who were long-term survivors, the level of CD18+ gene corrected leukocytes ranged from 0.72% to 8.37% at 1 |[ndash]| 1.5 years post-transplant. Although no adverse events have occurred during this period of observation, demonstration of polyclonality in the gene-corrected cells and characterization of the genomic sites of retroviral integration remain critical safety issues. To this end, we have demonstrated the polyclonality of contributing retrovirus-transduced cells in all six dogs using linear-amplification mediated PCR (LAM-PCR) with DNA obtained from peripheral blood leukocytes up to 18 months following infusion of gene-corrected cells. Multiple clones were shown to contribute to hematopoiesis; no predominant clones emerged over time. Retroviral insertion sites in four of the six dogs were identified using either LAM-PCR or ligation-mediated PCR (LM-PCR) followed by sequencing of the genomic sequence adjacent to the integrated viral LTR. Mapping of insertion sites was facilitated by the recent completion of the annotated dog genome sequence. To date, we have identified 60 unique retroviral insertion sites from four treated CLAD dogs. The insertions sites appear to be distributed almost equally across all 39 dog chromosomes, with a slight preference for chromosomes 2, 10 and 25. 43% of the sites were located within gene-coding regions of the genome (introns or exons). These observations match previous reports of preferential integration of MLV retroviral vectors near coding sequences. We are continuing to sequence and characterize a large number of retroviral insertion sites from the gene-corrected CLAD dogs. Analysis of these insertion sites should provide considerable insight into the risk of genotoxicity through insertional mutagenesis in a disease-specific large-animal model of a human hematopoietic disease.

Molecular defects in T- and B-cell primary immunodeficiency diseases

More than 120 inherited primary immunodeficiency diseases have been discovered in the past five decades, and the precise genetic defect in many of these diseases has now been identified. Increasing understanding of these molecular defects has considerably influenced both basic and translational research, and this has extended to many branches of medicine. Recent advances in both diagnosis and therapeutic modalities have allowed these defects to be identified earlier and to be more precisely defined, and they have also resulted in more promising long-term outcomes. The prospect of gene therapy continues to be included in the armamentarium of treatment considerations, because these conditions could be among the first to benefit from gene-therapy trials in humans.

The use of standard operating procedures in clinical research trials

Research nurses need to be familiar with standard operating procedure (SOP) documents. SOPs are distinct from nursing care plans. An SOP is an official, detailed, written instruction for the management of clinical trials; it ensures that all the functions and activities of a clinical trial are carried out consistently and efficiently. This article describes the background to, development and implementation of SOPs for a human gene therapy trial.

Propagation kinetics of retrovirus transgene vector and replication-competent retrovirus in static and microcarrier cell culture systems using different medium exchange strategies

While retrovirus vector (RV) is the main virus vector used in human gene therapy trials, the biosafety issues that surround currently used RVs have become a matter of concern. Similar to the insertional mutagenesis in the therapeutic target cells, the generation of replication-competent retrovirus (RCR) must be minimized during the manufacture of the virus vector. This work investigated the kinetics of RV and RCR production in PA317-RCM1 producer cells in static and microcarrier cell culture systems. RCR in the progeny of transduced Mus dunni cells was detected by the PCR method and the titer of RCR was quantified by cell-based S+/L− assay. The specific rates of RCR production in microcarrier cultures were 271–462% higher than those in the static well-plate cultures. Increased medium exchange operations yielded higher specific rates of RCR production in both static and microcarrier cultures. The optimal medium exchange strategy was on an every 2-day schedule, yielding the highest RV/RCR ratio in static culture but not microcarrier culture. Results of this study presented the difficulty for gene therapy processes that together with the product RV also unwanted RCR produced in two different cell culture systems.

Adenovirus vectors deleted for genes essential for viral DNA replication

Adenovirus (Ad) gene therapy vectors made replication defective by deletion of the E1 region (first-generation vectors) induce high-level inflammation that leads to loss of both transduced gene expression and transduced cells. First-generation vectors were initially considered to be incapable of viral DNA replication, but it is necessary to delete one or more of the genes, all in the E2 transcription unit, that encode proteins essential for Ad DNA replication to completely eliminate viral DNA replication. Vectors deleted for one or more of the E2 genes (second-generation vectors) induce reduced levels of inflammation in certain animal models and offer promise for understanding the mechanisms by which adenovirus vectors induce inflammation and how inflammation can be inhibited. While first-generation vectors dominated the initial human gene therapy trials using adenovirus vectors, second-generation vectors may offer greater promise and greater safety for clinical studies.