Donald B. Kohn

Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo

CCR5 is the major HIV-1 co-receptor, and individuals homozygous for a 32-bp deletion in CCR5 are resistant to infection by CCR5-tropic HIV-1. Using engineered zinc-finger nucleases (ZFNs), we disrupted CCR5 in human CD34+ hematopoietic stem/progenitor cells (HSPCs) at a mean frequency of 17% of the total alleles in a population. This procedure produces both mono- and bi-allelically disrupted cells. ZFN-treated HSPCs retained the ability to engraft NOD/SCID/IL2rγnull mice and gave rise to polyclonal multi-lineage progeny in which CCR5 was permanently disrupted. Control mice receiving untreated HSPCs and challenged with CCR5-tropic HIV-1 showed profound CD4+ T-cell loss. In contrast, mice transplanted with ZFN-modified HSPCs underwent rapid selection for CCR5−/− cells, had significantly lower HIV-1 levels and preserved human cells throughout their tissues. The demonstration that a minority of CCR5−/− HSPCs can populate an infected animal with HIV-1-resistant, CCR5−/− progeny supports the use of ZFN-modified autologous hematopoietic stem cells as a clinical approach to treating HIV-1.

Occurrence of leukaemia following gene therapy of X-linked SCID

Recombinant viral vectors have allowed gene transfer to be developed as a promising approach to the treatment of genetic diseases. Recently, gene therapy of children with X-linked severe combined immune deficiency resulted in impressive levels of immune reconstitution — a triumph that was later overshadowed by the development of leukaemia in two patients. What were the causes of this cancer, and how can the therapeutic benefits of gene therapy be achieved while minimizing risk to the patient?

Immune response to green fluorescent protein: implications for gene therapy.

Green fluorescent protein (GFP) is a widely used intracellular reporter molecule to assess gene transfer and expression. A potential use for GFP is as a co-expressed marker, to select and enrich gene-modified cells by flow cytometry. Processed peptides derived from GFP and presented by the major histocompatibility complex on the cell surface could potentially induce T cell immune responses against GFP+ cells. Thus, clinical application of GFP is premature, since in vivo studies on its immunogenicity are lacking. Therefore, we investigated immune responses against EGFP (enhanced-GFP) in two transplantable murine models: the BALB/c (H-2d) BM185 pre-B leukemia and the C57BL/6 (H-2b) EL-4 T cell lymphoma. BM185 and EL-4 cell lines modified to express high levels of EGFP showed drastic reduction of disease development when transplanted into immunocompetent mice. BM185/ EGFP did lead to rapid development of disease in immunodeficient Nu/Nu mice. Mice surviving BM185/EGFP leukemia challenge developed high cytotoxic T lymphocyte (CTL) responses against EGFP-expressing cells. Furthermore, immune stimulation against BM185/EGFP cells could also be induced by immunization with EGFP+ transduced dendritic cells. The effects of the co-expression of EGFP and immunomodulators (CD80 plus GM-CSF) were also investigated as an irradiated leukemia vaccine. EGFP co-expression by the vaccine did not interfere with the development of CTLs against the parental leukemia or with the anti-leukemia response in vivo. These results indicate that the immune response against EGFP may interfere with its applicability in gene insertion/replacement strategies but could potentially be employed for leukemia cell vaccines.

Newborn Screening for Severe Combined Immunodeficiency in 11 Screening Programs in the United States

IMPORTANCE Newborn screening for severe combined immunodeficiency (SCID) using assays to detect T-cell receptor excision circles (TRECs) began in Wisconsin in 2008, and SCID was added to the national recommended uniform panel for newborn screened disorders in 2010. Currently 23 states, the District of Columbia, and the Navajo Nation conduct population-wide newborn screening for SCID. The incidence of SCID is estimated at 1 in 100,000 births. OBJECTIVES To present data from a spectrum of SCID newborn screening programs, establish population-based incidence for SCID and other conditions with T-cell lymphopenia, and document early institution of effective treatments. DESIGN Epidemiological and retrospective observational study. SETTING Representatives in states conducting SCID newborn screening were invited to submit their SCID screening algorithms, test performance data, and deidentified clinical and laboratory information regarding infants screened and cases with nonnormal results. Infants born from the start of each participating program from January 2008 through the most recent evaluable date prior to July 2013 were included. Representatives from 10 states plus the Navajo Area Indian Health Service contributed data from 3,030,083 newborns screened with a TREC test. MAIN OUTCOMES AND MEASURES Infants with SCID and other diagnoses of T-cell lymphopenia were classified. Incidence and, where possible, etiologies were determined. Interventions and survival were tracked. RESULTS Screening detected 52 cases of typical SCID, leaky SCID, and Omenn syndrome, affecting 1 in 58,000 infants (95% CI, 1/46,000-1/80,000). Survival of SCID-affected infants through their diagnosis and immune reconstitution was 87% (45/52), 92% (45/49) for infants who received transplantation, enzyme replacement, and/or gene therapy. Additional interventions for SCID and non-SCID T-cell lymphopenia included immunoglobulin infusions, preventive antibiotics, and avoidance of live vaccines. Variations in definitions and follow-up practices influenced the rates of detection of non-SCID T-cell lymphopenia. CONCLUSIONS AND RELEVANCE Newborn screening in 11 programs in the United States identified SCID in 1 in 58,000 infants, with high survival. The usefulness of detection of non-SCID T-cell lymphopenias by the same screening remains to be determined.

Transplantation Outcomes for Severe Combined Immunodeficiency, 2000–2009

BACKGROUND The Primary Immune Deficiency Treatment Consortium was formed to analyze the results of hematopoietic-cell transplantation in children with severe combined immunodeficiency (SCID) and other primary immunodeficiencies. Factors associated with a good transplantation outcome need to be identified in order to design safer and more effective curative therapy, particularly for children with SCID diagnosed at birth. METHODS We collected data retrospectively from 240 infants with SCID who had received transplants at 25 centers during a 10-year period (2000 through 2009). RESULTS Survival at 5 years, freedom from immunoglobulin substitution, and CD3+ T-cell and IgA recovery were more likely among recipients of grafts from matched sibling donors than among recipients of grafts from alternative donors. However, the survival rate was high regardless of donor type among infants who received transplants at 3.5 months of age or younger (94%) and among older infants without prior infection (90%) or with infection that had resolved (82%). Among actively infected infants without a matched sibling donor, survival was best among recipients of haploidentical T-cell-depleted transplants in the absence of any pretransplantation conditioning. Among survivors, reduced-intensity or myeloablative pretransplantation conditioning was associated with an increased likelihood of a CD3+ T-cell count of more than 1000 per cubic millimeter, freedom from immunoglobulin substitution, and IgA recovery but did not significantly affect CD4+ T-cell recovery or recovery of phytohemagglutinin-induced T-cell proliferation. The genetic subtype of SCID affected the quality of CD3+ T-cell recovery but not survival. CONCLUSIONS Transplants from donors other than matched siblings were associated with excellent survival among infants with SCID identified before the onset of infection. All available graft sources are expected to lead to excellent survival among asymptomatic infants. (Funded by the National Institute of Allergy and Infectious Diseases and others.).

Genetic therapies against HIV

Highly active antiretroviral therapy prolongs the life of HIV-infected individuals, but it requires lifelong treatment and results in cumulative toxicities and viral-escape mutants. Gene therapy offers the promise of preventing progressive HIV infection by sustained interference with viral replication in the absence of chronic chemotherapy. Gene-targeting strategies are being developed with RNA-based agents, such as ribozymes, antisense, RNA aptamers and small interfering RNA, and protein-based agents, such as the mutant HIV Rev protein M10, fusion inhibitors and zinc-finger nucleases. Recent advances in T-cell–based strategies include gene-modified HIV-resistant T cells, lentiviral gene delivery, CD8+ T cells, T bodies and engineered T-cell receptors. HIV-resistant hematopoietic stem cells have the potential to protect all cell types susceptible to HIV infection. The emergence of viral resistance can be addressed by therapies that use combinations of genetic agents and that inhibit both viral and host targets. Many of these strategies are being tested in ongoing and planned clinical trials.

Redirecting specificity of T-cell populations for CD19 using the Sleeping Beauty system.

Genetic modification of clinical-grade T cells is undertaken to augment function, including redirecting specificity for desired antigen. We and others have introduced a chimeric antigen receptor (CAR) to enable T cells to recognize lineage-specific tumor antigen, such as CD19, and early-phase human trials are currently assessing safety and feasibility. However, a significant barrier to next-generation clinical studies is developing a suitable CAR expression vector capable of genetically modifying a broad population of T cells. Transduction of T cells is relatively efficient but it requires specialized manufacture of expensive clinical grade recombinant virus. Electrotransfer of naked DNA plasmid offers a cost-effective alternative approach, but the inefficiency of transgene integration mandates ex vivo selection under cytocidal concentrations of drug to enforce expression of selection genes to achieve clinically meaningful numbers of CAR(+) T cells. We report a new approach to efficiently generating T cells with redirected specificity, introducing DNA plasmids from the Sleeping Beauty transposon/transposase system to directly express a CD19-specific CAR in memory and effector T cells without drug selection. When coupled with numerical expansion on CD19(+) artificial antigen-presenting cells, this gene transfer method results in rapid outgrowth of CD4(+) and CD8(+) T cells expressing CAR to redirect specificity for CD19(+) tumor cells.

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.).

American Society of Gene Therapy (ASGT) Ad Hoc Subcommittee on Retroviral-Mediated Gene Transfer to Hematopoietic Stem Cells

Gene transfer using retroviral vectors has been under clinical study for more than 12 years1. Many studies have targeted hematopoietic stem cells (HSCs) as a potentially enduring and renewable source of gene-modified blood cells for the treatment of specific genetic diseases, cancer, leukemia, and HIV-1 infection2. Although initial studies were hampered by very low levels of gene transfer to HSCs, incremental progress has been realized in the efficiency of gene transfer to HSCs. These advances have culminated in the report of clinically significant restoration of immunity in patients with the X-linked form of severe combined immune deficiency (XSCID) by Alain Fischer, Marina Cavazzana-Calvo, and colleagues at the Hopital Necker Enfants Malades in Paris3. Their study and those conducted by Adrian Thrasher and colleagues at the Great Ormond Street Childrens Hospital in London for XSCID and by Claudio Bordignon and colleagues at the Hospital San Raffaele in Milan for children with SCID due to deficiency of adenosine deaminase (ADA) provide incontrovertible proof that gene therapy can ameliorate genetic diseases4,5.

Clonality analysis after retroviral-mediated gene transfer to CD34+ cells from the cord blood of ADA-deficient SCID neonates

A clinical trial of retroviral-mediated transfer of the adenosine deaminase (ADA) gene into umbilical cord blood CD34+ cells was started in 1993. ADA-containing peripheral blood mononuclear cells (PBMCs) have persisted in patients from this trial, with T lymphocytes showing the highest prevalence of gene marking. To gain a greater understanding of the nature and number of the transduced cells that were engrafted, we used linear amplification–mediated PCR (LAM-PCR) to identify clonal vector proviral integrants. In one patient, a single vector integrant was predominant in T lymphocytes at a stable level over most of the eight-year time span analyzed and was also detected in some myeloid samples. T-cell clones with the predominant integrant, isolated after eight years, showed multiple patterns of T-cell receptor (TCR) gene rearrangement, indicating that a single pre-thymic stem or progenitor cell served as the source of the majority of the gene-marked cells over an extended period of time. It is important to distinguish the stable pattern of monoclonal gene marking that we observed here from the progressive increase of a T-cell clone with monoclonal gene marking that results from leukemic transformation, as observed in two subjects in a clinical trial of gene therapy for X-linked severe combined immunodeficiency (SCID).