Kit L. Shaw

A Modified γ-Retrovirus Vector for X-Linked Severe Combined Immunodeficiency

BACKGROUND In previous clinical trials involving children with X-linked severe combined immunodeficiency (SCID-X1), a Moloney murine leukemia virus-based γ-retrovirus vector expressing interleukin-2 receptor γ-chain (γc) complementary DNA successfully restored immunity in most patients but resulted in vector-induced leukemia through enhancer-mediated mutagenesis in 25% of patients. We assessed the efficacy and safety of a self-inactivating retrovirus for the treatment of SCID-X1. METHODS We enrolled nine boys with SCID-X1 in parallel trials in Europe and the United States to evaluate treatment with a self-inactivating (SIN) γ-retrovirus vector containing deletions in viral enhancer sequences expressing γc (SIN-γc). RESULTS All patients received bone marrow-derived CD34+ cells transduced with the SIN-γc vector, without preparative conditioning. After 12.1 to 38.7 months of follow-up, eight of the nine children were still alive. One patient died from an overwhelming adenoviral infection before reconstitution with genetically modified T cells. Of the remaining eight patients, seven had recovery of peripheral-blood T cells that were functional and led to resolution of infections. The patients remained healthy thereafter. The kinetics of CD3+ T-cell recovery was not significantly different from that observed in previous trials. Assessment of insertion sites in peripheral blood from patients in the current trial as compared with those in previous trials revealed significantly less clustering of insertion sites within LMO2, MECOM, and other lymphoid proto-oncogenes in our patients. CONCLUSIONS This modified γ-retrovirus vector was found to retain efficacy in the treatment of SCID-X1. The long-term effect of this therapy on leukemogenesis remains unknown. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01410019, NCT01175239, and NCT01129544.).

Myeloid dysplasia and bone marrow hypocellularity in adenosine deaminase-deficient severe combined immune deficiency

Genetic deficiency of adenosine deaminase (ADA) can cause profound lymphopenia and result in the clinical presentation of severe combined immune deficiency (SCID). However, because of the ubiquitous expression of ADA, ADA-deficient patients often present also with nonimmunologic clinical problems, affecting the skeletal, central nervous, endocrine, and gastrointestinal systems. We now report that myeloid dysplasia features and bone marrow hypocellularity are often found in patients with ADA-SCID. As a clinical correlate to this finding, we have observed vulnerability to antibiotic-induced myelotoxicity and prolonged neutropenia after nonmyeloablative chemotherapy. We have also noted that, in the absence of enzyme replacement therapy, absolute neutrophil counts of patients with ADA deficiency vary inversely with the accumulation of deoxynucleotides. These data have significant implications for the application of standard and investigational therapies to patients with ADA-SCID and support further studies to investigate the possibility that ADA deficiency is associated with a stem cell defect. These trials were registered at www.clinicaltrials.gov as #NCT00018018 and #NCT00006319.

A Tale of Two SCIDs

Gene therapy for severe combined immunodeficiency: Do the benefits outweigh the risks? Hematopoietic stem cell (HSC) transplantation may be curative for severe combined immunodeficiency (SCID). However, for a majority of infants with SCID a suitable donor is not available, and even with a matched donor, allogeneic HSC transplantation itself carries potential complications such as graft-versus-host disease as well as side effects from myelosuppressive chemotherapy. In the past decade, substantial advances have been made in the transplantation of gene-modified autologous HSCs, especially for two forms of SCID: X-linked SCID (SCID-X1) and adenosine deaminase (ADA)–deficient SCID. Two new reports in this issue of Science Translational Medicine add to the accumulating findings from gene therapy trials in Italy, France, and the United States that show clinical benefits of this alternative treatment.

Clinical efficacy of gene-modified stem cells in adenosine deaminase–deficient immunodeficiency

BACKGROUND. Autologous hematopoietic stem cell transplantation (HSCT) of gene-modified cells is an alternative to enzyme replacement therapy (ERT) and allogeneic HSCT that has shown clinical benefit for adenosine deaminase–deficient (ADA-deficient) SCID when combined with reduced intensity conditioning (RIC) and ERT cessation. Clinical safety and therapeutic efficacy were evaluated in a phase II study. METHODS. Ten subjects with confirmed ADA-deficient SCID and no available matched sibling or family donor were enrolled between 2009 and 2012 and received transplantation with autologous hematopoietic CD34+ cells that were modified with the human ADA cDNA (MND-ADA) &ggr;-retroviral vector after conditioning with busulfan (90 mg/m2) and ERT cessation. Subjects were followed from 33 to 84 months at the time of data analysis. Safety of the procedure was assessed by recording the number of adverse events. Efficacy was assessed by measuring engraftment of gene-modified hematopoietic stem/progenitor cells, ADA gene expression, and immune reconstitution. RESULTS. With the exception of the oldest subject (15 years old at enrollment), all subjects remained off ERT with normalized peripheral blood mononuclear cell (PBMC) ADA activity, improved lymphocyte numbers, and normal proliferative responses to mitogens. Three of nine subjects were able to discontinue intravenous immunoglobulin replacement therapy. The MND-ADA vector was persistently detected in PBMCs (vector copy number [VCN] = 0.1–2.6) and granulocytes (VCN = 0.01–0.3) through the most recent visits at the time of this writing. No patient has developed a leukoproliferative disorder or other vector-related clinical complication since transplant. CONCLUSION. These results demonstrate clinical therapeutic efficacy from gene therapy for ADA-deficient SCID, with an excellent clinical safety profile. TRIAL REGISTRATION. ClinicalTrials.gov NCT00794508. FUNDING. Food and Drug Administration Office of Orphan Product Development award, RO1 FD003005; NHLBI awards, PO1 HL73104 and Z01 HG000122; UCLA Clinical and Translational Science Institute awards, UL1RR033176 and UL1TR000124.

C-8. Immunological and Metabolic Correction After Lentiviral Vector Gene Therapy for ADA Deficiency

Background: Adenosine deaminase deficiency leads to severe combined immunodeficiency. Autologous haematopoietic stem cell gene therapy may offer a curative therapy. We developed a self inactivating lentiviral vector in which the human ADA gene is driven by an internal EFS promoter. Parallel trials using this vector were conducted in UCL, London and UCLA, Los AngelesObjective: A Phase I/II trial to assess the safety and efficacy of EFS-ADA lentiviral vector mediated gene modification of autologous CD34+ cells from ADA-deficient individuals.Methods: 20 patients (12 male; 8 female) aged between 0.4-6.5yrs were treated. All had been on enzyme replacement prior to treatment. CD34+ cells were collected either by BM harvest or peripheral blood stem cell mobilisation. Busulfan i.v. at a single dose of 4-5mg/kg was given as conditioning. CD34+ cells were stimulated with cytokines for 24hrs before transduction with vector for a further 18hrs. The dose of CD34+ cells returned ranged from 3-17 × 10e6/kg with a vector copy number (VCN) in the transduced population of between 0.25-6.3 vector copies per cell.Results: The procedure was well tolerated by all patients with no adverse events related to Busulfan conditioning or the vector. The follow up in all patients ranges from 1-35 months. All 20 patients remain off PEG ADA enzyme replacement therapy. There is evidence of immunological and metabolic recovery in all 16 patients treated for longer than 6 months. All patients have shown a rise in total T cell counts and at last follow-up the following levels were seen; total T cells, range 220-3,370; CD4+ cells 190-1509. Mitogen responses to PHA have increased in all patients and there is naive T cell recovery with an increase in CD45RA+CD4+CD27+ subpopulations and the number of TRECs. Immunoglobulin replacement therapy has been stopped in 4 patients so far and the one patient who has completed vaccinations shows normal vaccine specific responses. Gene marking is detectable in the periphery with highest marking in T cells but significant marking in B cells, NK cells and myeloid cells. Intracellular red blood cell ADA activity was negligible prior to gene therapy but at latest follow up is detectable at near or above normal levels in all patients. Integration site analysis shows occasional expansions but no persistence of expanded clones. There is no detection of expansion of clones with genes previously associated with insertional mutagenesis. All 20 patients are clinically well and the earliest treated patients are free of social restriction.Summary: Lentiviral vector mediated gene therapy for ADA deficiency is well tolerated and allows effective recovery of immunological and metabolic parameters with no evidence for mutagenesis thus far. These trials of 20 patients in two centres demonstrates the high efficacy and safety of this treatment strategy.

Cytoreductive conditioning intensity predicts clonal diversity in ADA-SCID retroviral gene therapy patients

Retroviral gene therapy has proved efficacious for multiple genetic diseases of the hematopoietic system, but roughly half of clinical gene therapy trial protocols using gammaretroviral vectors have reported leukemias in some of the patients treated. In dramatic contrast, 39 adenosine deaminase-deficient severe combined immunodeficiency (ADA-SCID) patients have been treated with 4 distinct gammaretroviral vectors without oncogenic consequence. We investigated clonal dynamics and diversity in a cohort of 15 ADA-SCID children treated with gammaretroviral vectors and found clear evidence of genotoxicity, indicated by numerous common integration sites near proto-oncogenes and by increased abundance of clones with integrations near MECOM and LMO2 These clones showed stable behavior over multiple years and never expanded to the point of dominance or dysplasia. One patient developed a benign clonal dominance that could not be attributed to insertional mutagenesis and instead likely resulted from expansion of a transduced natural killer clone in response to chronic Epstein-Barr virus viremia. Clonal diversity and T-cell repertoire, measured by vector integration site sequencing and T-cell receptor β-chain rearrangement sequencing, correlated significantly with the amount of busulfan preconditioning delivered to patients and to CD34+ cell dose. These data, in combination with results of other ADA-SCID gene therapy trials, suggest that disease background may be a crucial factor in leukemogenic potential of retroviral gene therapy and underscore the importance of cytoreductive conditioning in this type of gene therapy approach.

Regulated Expansion of Human Pancreatic β-Cells

Although pancreatic β-cell transplantation may serve as a potential cure for diabetes mellitus (DM), limited donor tissue availability poses a major challenge. Thus, there is a great demand to find new sources for pancreatic β-cells. Here, we present a lentiviral vector-based approach to achieve β-cell proliferation through the β-cell-specific activation of the hepatocyte growth factor (HGF)/cmet signaling pathway. The methodology is based on the β-cell-specific expression of a ligand-inducible, chimeric receptor (F36Vcmet), under transcriptional control of the promoter from the human insulin gene, and its ability to induce HGF/cmet signaling in the presence of a synthetic ligand (AP20187). High transduction efficiency of human pancreatic islets was achieved utilizing this approach with chimeric receptor expression confined to the β-cell population. In addition, specific proliferation of human pancreatic β-cells was induced utilizing this approach. Selective, regulated β-cell expansion may help to provide greater availability of cells for transplantation in patients with DM.Although pancreatic beta-cell transplantation may serve as a potential cure for diabetes mellitus (DM), limited donor tissue availability poses a major challenge. Thus, there is a great demand to find new sources for pancreatic beta-cells. Here, we present a lentiviral vector-based approach to achieve beta-cell proliferation through the beta-cell-specific activation of the hepatocyte growth factor (HGF)/cmet signaling pathway. The methodology is based on the beta-cell-specific expression of a ligand-inducible, chimeric receptor (F36Vcmet), under transcriptional control of the promoter from the human insulin gene, and its ability to induce HGF/cmet signaling in the presence of a synthetic ligand (AP20187). High transduction efficiency of human pancreatic islets was achieved utilizing this approach with chimeric receptor expression confined to the beta-cell population. In addition, specific proliferation of human pancreatic beta-cells was induced utilizing this approach. Selective, regulated beta-cell expansion may help to provide greater availability of cells for transplantation in patients with DM.

30. Phase II Clinical Trial of Gene Therapy for Adenosine Deaminase-Deficient Severe Combined Immune Deficiency (ADA-SCID) Using a γ-Retroviral Vector

We report follow-up of subjects treated in a Phase II study of gene therapy for ADA-SCID. Between 2009 and 2012, ten ADA-deficient SCID patients were treated by γ-retroviral-mediated gene transfer (MND-ADA) to their bone marrow CD34+ cells. The subjects were given non-myeloablative chemotherapy (busulfan @ 90 mg/m2) and were withdrawn from PEG-ADA enzyme replacement therapy (ERT) prior to infusion of autologous gene-modified cells. Subject age at the time of treatment ranged from 3 months to 15 years (median = 11.5 months). Follow-up times range from 2 to 5 years. All but one subject, who was 15-years old at the time of treatment, remain off PEG-ADA ERT with immune reconstitution that reached maximal level between 6 and 12 months after transplant and was maintained thereafter. Vector marking in peripheral blood cells remained consistently detectable (> 0.1 copy/PBMC and ≥ 0.003 copy/granulocyte) at 2 years and later after transplant in subjects who discontinued ERT. These subjects also had PBMC ADA enzymatic activity in the normal range and red blood cell deoxynucleotide levels below 10%. Three subjects have discontinued intravenous immunoglobulin; five subjects have discontinued prophylactic antibiotics. All subjects have polyclonal gene marking with no sign of lymphoproliferative disease. The subjects remain in good health without infections or other complications.

240. Stable and Clinically Benign Clonal Dominance in an ADA-SCID Patient Treated With Retroviral Gene Therapy

To date, adverse events involving genotoxicity and oncogenic transformation have occurred in several gene therapy trials using oncoretroviral vectors. In striking and surprising contrast, insertional oncogenesis has not been observed in multiple trials treating SCID caused by adenosine deaminase deficiency (ADA-SCID), despite the use of very similar oncoretroviral vectors. In our trial for ADA-SCID, we employed non-restrictive LAM-PCR to monitor clonality and observed that one patient developed a marked clonal dominance that was stable over multiple years. Despite this, the patient has been clinically well up to present. The dominant clone was confirmed using an integration site-specific digital PCR assay, and it accounted for up to 85% of vector integrations in peripheral blood mononuclear cell (PBMC) DNA. The vector integration in the dominant clone is in a gene-poor region of chromosome 21, which is also a common insertion site observed in our cohort of patients. RNA-seq expression analysis of cells of this clone did not detect any dysregulation of nearby genes. Importantly, this analysis would have also detected unannotated non-coding RNAs or novel transcripts being produced from the vector, and we observed neither. The majority of vector copies detected in PBMC DNA were contributed by the NK lineage, and the clone was most dominant in NK cells. Compared to healthy donor NK cells, the patients NK cells more highly expressed the NK receptor NKG2C. The beginning of the clonal expansion coincides with the onset of Epstein-Barr virus (EBV) viremia in the patient, which also correlates with the expansion of a potential EBV-reactive T-cell clone, as indicated by flow cytometric TCR beta family analysis. We hypothesize that NK cells expressing NKG2C and bearing the chr21 IS expanded in response to the EBV infection, which was not fully controlled by the patients adaptive immune response.