X-linked Severe Combined Immunodeficiency Syndrome Research Articles

Malignant transformation of mature T cells after gammaretrovirus mediated transfer of nucleophosmin-anaplastic lymphoma kinase oncogene.

Gene therapy has been in use to cure hereditary and acquired diseases by incorporating the desired gene into the cells with the help of gammaretroviral vectors. Despite the success of this therapy in X-linked severe combined immunodeficiency syndrome, few patients developed leukemia as a major adverse event due to retroviral insertional mutagenesis within stem cells. In experimental animals also, retroviral-mediated gene transfer technique resulted in the development of leukemia. On the other hand, evidence suggests that mature T cells (TC) are relatively resistant to transformation even after retroviral-mediated transfer of potent oncogenes Tcl1, ΔTrkA and LMO2 with no reported side effects yet. To further address the safety issue for TC use in gene therapy, this study investigated susceptibility of mature polyclonal TC to malignant transformation by the retroviral-mediated transfer of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) oncogene. Wild-type mature TC, isolated from C57BL/6 donor mice (genetic background Ly5.1) were transduced with gamma-retroviral vectors encoding the potent TC oncogene NPM-ALK or the control vector enhanced green fluorescent protein eGFP. The cells were then transplanted into RAG-1 deficient recipient mice (genetic background Ly5.2). Two out of five mice from NPM-ALK oncogene group developed leukemia/lymphoma after latency periods (153 and 250 days, respectively). None of the mice from the control group developed any malignancy throughout the observational period. Mature polyclonal TC are relatively susceptible to malignant transformation after gamma-retroviral mediated transfer of NPM-ALK oncogene; hence safety of TC use in gene therapy should be further investigated to avoid the possible side-effect of development of leukemia/lymphoma.

In utero haematopoietic stem cell transplantation – experimental progress towards clinical application

In utero haematopoietic stem cell transplantation (IUHCT) is a potential therapeutic alternative to postnatal allogeneic bone marrow transplantation (BMT) for congenital haematologic disorders that can be diagnosed early in gestation and can be cured by BMT. The rationale is to take advantage of normal events during haematopoietic and immunologic ontogeny to facilitate allogeneic haematopoietic engraftment. The most important of these is the normal process of thymic processing of self-antigen to create a state of self-tolerance. Introduction of allogeneic antigen in the form of haematopoietic stem cells results in a state of permanent donor specific tolerance, eliminating the need for immunosuppression. Although the rationale remains compelling, IUHCT has only been clinically successful in X-linked severe combined immunodeficiency syndrome, a disease in which a competitive advantage exists for donor lymphoid cells. In other disorders, such as the haemoglobinopathies, where host-cell competition is a major barrier, IUHCT has not yet succeeded. However, great experimental progress has recently been made in pre-clinical animal models. Strategies based on prenatal tolerance induction to facilitate post-natal non-toxic cellular transplantation appear promising and clinical application is likely imminent. Because donor specific tolerance induction requires relatively minimal engraftment, this strategy may hold the key to broad clinical application of IUHCT in the near future.

Integration-Mediated Activation of PRDM16 and HMGA2 in Multiple Clones without Adverse Hematopoietic Consequences Following Transplant of Autologous MGMTP140K Gene-Modified CD34+ Cells

Abstract 2053▪▪This icon denotes a clinically relevant abstractDrug resistance gene therapy with mutant MGMTP140K gene-modified hematopoietic CD34+ cells has been proposed to circumvent myelosuppression associated with alkylating agent chemotherapy; however, the safety of this approach has not yet been demonstrated. We have achieved successful engraftment of MGMTP140K gene-modified hematopoietic progenitor cells in 3 glioblastoma patients following BCNU (600mg/m2) conditioning, as part of a Phase I/II clinical study which includes post-transplant combination O6-benzylguanine (O6BG) and temozolomide (TMZ) chemotherapy. Two of these patients, one of whom remains alive with no evidence for disease progression at more than 2 years since diagnosis, received a total of 9 and 4 cycles of O6BG/TMZ chemotherapy, respectively. We observed transient increases in the number of circulating gene-modified white blood cells (WBCs) and granulocytes in these patients following each cycle. Analysis of CD34+ colony forming cells (CFCs) in peripheral blood revealed increases in the number of gene-modified CFCs over time and with multiple cycles of chemotherapy. Given this, we have conducted a longitudinal retroviral integration site (RIS) analysis in an attempt to identify any selective advantage for gene-modified clones following repeated O6BG/TMZ regimens. In the first patient, at day 200 following transplant, 2 separate RISs were identified, each mapping to the intronic region between exons 1 and 2 of the human PRDM16 gene, previously identified as being associated with insertional activation and clonal expansion in a gene therapy trial for X-linked chronic granulomatous disease (Ott et al 2006). Semi-quantitative capture site frequency analysis revealed that these 2 clones with PRDM16 integrations constituted >20% of the gene-modified cell pool, which included hundreds of RISs at this time point in this patient. Overall gene marking at this same time point was 39.9% of peripheral blood WBCs, indicating that as many as 8% of circulating WBCs arose from these two clones. In the second patient, 3 distinct RISs were identified mapping to the 3’untranslated region (UTR) of the HMGA2 gene, within a cluster of let-7 microRNA binding domains known to be responsible for post-transcriptional regulation of this gene product. While HMGA2-associated RISs have been reported in 2 other gene therapy trials for β-thalassemia (Cavazzana-Calvo et al 2010) and X-linked severe combined immunodeficiency syndrome (Wang et al 2010), the genomic loci of these RISs mapped to the third intron of this genomic sequence. Capture frequency analysis indicated that overall contribution of the most abundant HMGA2-associated clone identified in this study reached 5.2% of gene-modified peripheral blood WBCs at day 300 after transplant. Analysis of PRDM16 and HMGA2 transcripts in each patient’s cells by reverse-transcriptase real-time PCR indicated increased transcription of these genes in peripheral blood and bone marrow WBCs. Analysis of WBC subsets in the first patient found PRDM16 RISs present in granulocyte and CD3+ blood cell lineages, suggesting these clones originated from multipotential gene-modified hematopoietic stem cells capable of differentiating despite PRDM16 transactivation. Furthermore, since discontinuation of chemotherapy, we have observed a steady decline in levels of both PRDM16 clones, with complete disappearance of one clone. Extended analysis of HMGA2 expression and clone tracking in WBC subsets of the second patient is currently under way. Importantly, analysis of both patients bone marrow demonstrated normal cytogenetics, with no evidence for MDS or leukemia by hematopathology and flow cytometry. We believe that these data, in combination with other clinical gene therapy studies identifying similar clones, indicate the PRDM16 and HMGA2 gene sequences as “hot-spots” for either initial retrovirus integration in CD34+ hematopoietic progenitor and stem cells, or as genomic loci where aberrant gene expression, as a result of retrovirus integration, is associated with increased cell proliferation. At this time, these observations suggest that in the absence of underlying genomic disease, in vivo fluctuations in PRDM16- and HMGA2-associated clones following transplant may be a common, clinically inconsequential phenomenon in retrovirus-mediated gene therapy targeting CD34+ cell populations for modification. Disclosures:No relevant conflicts of interest to declare.

RNA silencing and HIV: A hypothesis for the etiology of the severe combined immunodeficiency induced by the virus

A novel intrinsic HIV-1 antisense gene was previously described with RNA initiating from the region of an HIV-1 antisense initiator promoter element (HIVaINR). The antisense RNA is exactly complementary to HIV-1 sense RNA and capable of forming ~400 base-pair (bp) duplex RNA in the region of the long terminal repeat (LTR) spanning the beginning portion of TAR in the repeat (R) region and extending through the U3 region. Duplex or double-stranded RNA of several hundred nucleotides in length is a key initiating element of RNA interference (RNAi) in several species. This HIVaINR antisense RNA is also capable of forming multiple stem-loop or hairpin-like secondary structures by M-fold analysis, with at least one that perfectly fits the criteria for a microRNA (miRNA) precursor. MicroRNAs (miRNAs) interact in a sequence-specific manner with target messenger RNAs (mRNAs) to induce either cleavage of the message or impede translation. Human mRNA targets of the predicted HIVaINR antisense RNA (HAA) microRNAs include mRNA for the human interleukin-2 receptor gamma chain (IL-2RG), also called the common gamma (γc) receptor chain, because it is an integral part of 6 receptors mediating interleukin signalling (IL-2R, IL-4R, IL-7R, IL-9R, IL-15R and IL-21R). Other potential human mRNA targets include interleukin-15 (IL-15) mRNA, the fragile × mental retardation protein (FMRP) mRNA, and the IL-1 receptor-associated kinase 1 (IRAK1) mRNA, amongst others. Thus the proposed intrinsic HIVaINR antisense RNA microRNAs (HAAmiRNAs) of the human immunodeficiency virus form complementary targets with mRNAs of a key human gene in adaptive immunity, the IL-2Rγc, in which genetic defects are known to cause an X-linked severe combined immunodeficiency syndrome (X-SCID), as well as mRNAs of genes important in innate immunity. A new model of intrinsic RNA silencing induced by the HIVaINR antisense RNA in the absence of Tat is proposed, with elements suggestive of both small interfering RNA (siRNA) and miRNA.

Long-Term Clinical and Molecular Follow-up of Large Animals Receiving Retrovirally Transduced Stem and Progenitor Cells: No Progression to Clonal Hematopoiesis or Leukemia

There has been significant progress toward clinically relevant levels of retroviral gene transfer into hematopoietic stem cells (HSC), and the therapeutic potential of HSC-based gene transfer has been convincingly demonstrated in children with severe combined immunodeficiency syndrome (SCID). However, the subsequent development of leukemia in two children with X-linked SCID who were apparently cured after transplantation of retrovirally corrected CD34+ cells has raised concerns regarding the safety of gene therapy approaches utilizing integrating vectors. Nonhuman primates and dogs represent the best available models for gene transfer safety and efficacy and are particularly valuable for evaluation of long-term effects. We have followed 42 rhesus macaques, 23 baboons, and 17 dogs with significant levels of gene transfer for a median of 3.5 years (range 1–7) after infusion of CD34+ cells transduced with retroviral vectors expressing marker or drug-resistance genes. None developed abnormal hematopoiesis or leukemia. Integration site analysis confirmed stable, polyclonal retrovirally marked hematopoiesis, without progression toward mono- or oligoclonality over time. These results suggest that retroviral integrations using replication-incompetent vectors, at copy numbers achieved using standard protocols, are unlikely to result in leukemogenesis and that patient- or transgene-specific factors most likely contributed to the occurrence of leukemia in the X-SCID gene therapy trial.

X-linked severe combined immunodeficiency syndrome: the first Korean case with gamma c chain gene mutation and subsequent genetic counseling.

X-linked severe combined immunodeficiency (X-SCID) is a rare, life-threatening immune disorder, caused by mutations in the γc chain gene, which encodes an essential component of the cytokine receptors for interleukin-2 (IL-2), IL-4, IL-7, IL-9, IL-15, and IL-21. A 13-month-old boy with recurrent infections who had reduced serum immunoglobulin levels and decreased numbers of CD3, CD16/56 cells was evaluated for γc chain gene mutation and protein expression. The patient had a C-to-T point mutation at nucleotide position 690, one of the hot spots, resulting in a single amino acid substitution of cysteine for arginine (R226C), as determined by direct sequencing and PCR-RFLP. The patient's mother was a heterozygous carrier. Percutaneous umbilical cord blood sampling was performed at the 6-month of gestation in a subsequent pregnancy. As the immunophenotype of the fetus showed an identical pattern, the pregnancy was terminated and genetic analysis of the abortus confirmed recurrence. This is the first report of the molecular diagnosis of X-SCID in Korea. Genetic analysis of the γc chain gene is useful for definite diagnosis and genetic counseling for X-SCID.

Human hematopoietic cell express two forms of the cytokine receptor common gamma-chain (gamma c).

Recent studies have revealed that the gamma-chain of the IL-2 receptor is shared by the receptors for IL-4, IL-7, IL-9, IL-13, and IL-15, and it is therefore also referred to as the common gamma-chain (gamma c). Mutations of gamma c result in X-linked severe combined immunodeficiency syndrome in humans, indicating that gamma c is essential for normal development and function of the immune system. We demonstrate that human hematopoietic cells express two gamma c transcripts differing in their carboxyl terminal coding region. One transcript is the previously reported sequence (gamma c-long), whereas the newly identified sequence exhibits a deletion of 72 nucleotides close to the 3'-end of the open reading frame (gamma c-short). This alteration predicts a loss of 24 amino acids including a conserved tyrosine residue which is shared by several members of the cytokine receptor family. The presence of these two distinct forms of gamma c transcripts was demonstrated by sequencing of reversely transcribed and polymerase chain reaction (RT-PCR) amplified mRNA, restriction digestion of the RT-PCR products, RNAse protection, and Northern blotting from human cell lines and human peripheral blood lymphocytes. Furthermore, the two variants were present in peripheral blood lymphocytes from both female and male donors, which rules out allelic variants since gamma c is a single copy gene located on the X chromosome. A truncation mutant at a site near the observed changes in gamma c-short has been reported by others to alter biochemical events activated by cytokines. This combined with the loss of a potential SH2 "docking" site in gamma c-short suggests that gamma c-long and gamma c-short may link to different signaling pathways and may play an important role in determining the cellular response to IL-2, IL-4, IL-7, IL-9, IL-13, IL-15.

Retroviral Vector for Gene Therapy of X-Linked Severe Combined Immunodeficiency Syndrome

X-linked severe combined immunodeficiency syndrome (X-SCID) is a genetic disorder characterized by profound impairment of cell-mediated and humoral immunity. Affected children die of recurrent infections within 2 years of birth unless rescued by allogeneic transplantation from a suitable donor. Recently, the genetic defect responsible for X-linked SCID has been identified as a mutation in the gamma chain of the IL-2 receptor, a protein also shared by the IL-4 and IL-7 receptors and therefore now denoted the common gamma chain (gamma c). We report here the development of a high-titer amphotropic retroviral vector for transfer of gamma c. This vector was used to transfer a copy of the gamma c cDNA to murine 3T3 fibroblasts, CD34-enriched hematopoietic progenitor cells obtained from bone marrow and umbilical cord blood of normal donors, and to transplanted murine bone marrow progenitors. Murine 3T3 cells transduced by the retroviral vector were analyzed by Southern blot hybridization and Western transfer. Southern analysis confirmed the integration of unrearranged proviral DNA, and Western blot analysis demonstrated the expression of gamma c protein. CD34-enriched cells were infected with viral vectors bearing gamma c and grown in methylcellulose media. Individual colonies and pools of cells were analyzed 2 weeks later by polymerase chain reaction assay, which confirmed the proviral marking. The vector was also used to transfer a copy of the gamma c cDNA to murine bone marrow cells in a transplantation model. Infected marrow was transplanted into syngeneic Balb/c mice.(ABSTRACT TRUNCATED AT 250 WORDS)