A self-inactivating lentiviral vector for SCID-X1 gene therapy that does not activate LMO2 expression in human T cells

Abstract

IntroductionX-linked severe combined immunodeficiency (SCID-X1) is caused by loss-of-function mutations in the ␥ c gene (also known as IL2RG), 1,2 which encodes the common ␥-chain (␥ c ) subunit for the IL2, IL4, IL7, IL9, IL15, and IL21 receptors and is required for signal transduction with these cytokines. 3 Human SCID-X1 is characterized by lack of T, natural killer (NK) cells, and nonfunctional B cells and is fatal early in life from progressive infections if left untreated. Allogeneic stem cell transplantation is the current standard of care and can be curative, particularly when a matched sibling donor is available. 4 However, most patients lack a matched sibling donor and therefore receive transplants using haploidentical grafts obtained from a parent. These patients do significantly less well than those receiving matched sibling transplants, both in terms of overall survival and in terms of persistent immunodeficiency. [5][6][7] Most experts agree that better treatment is desirable for SCID-X1 patients who lack a matched sibling donor. [8][9][10] Gene therapy is one of several approaches that is being developed as an alternative to haploidentical allogeneic transplantation. [11][12][13][14][15] Pioneering studies done in Europe using vectors derived from the mouse Moloney leukemia virus (MLV) have proven that this approach can be effective because of restoration of T-cell numbers and function as well as humoral immunity. [11][12][13][14] However, the development of T-cell leukemia in 5 of 20 of these patients has uncovered a serious, unanticipated side effect associated with these gene therapy vectors. [16][17][18] Comprehensive molecular analyses of the leukemia cases have identified vector insertions into a small number of T-cell proto-oncogenes, most particularly LMO2, leading to its aberrant and high-level expression. [16][17][18] Further analysis of these integration sites has revealed that most, if not all, cases of gene activation were the result of the strong enhancer elements located in the U3 region of the MLV long-terminal repeat (LTR). Clearly, new vectors for SCID-X1 should not contain these MLV LTR enhancer elements to achieve better safety. This can be accomplished using self-inactivating (SIN) vectors that contain deletions in the U3 region of the LTR and is possible using either MLV or HIV-based vectors.We chose to use lentiviral vectors because they are more efficient for transducing quiescent human hematopoietic stem cells (HSCs), 19 and a recent study has suggested that, when an internal LTR enhancer is present, SIN lentiviral vectors require 10-fold higher copy numbers than an SIN-MLV vector to cause tumors in a tumor-prone mouse model, 20 presumably because of the known differences in integration site profile between HIV versus MLV vectors. 21 Another important consideration is the recent development of a stable packaging system for producing SIN lentiviral vectors. 22 This eliminates the need for using transient transfection methods with multiple plasmid DNAs to produce lentiviral...

Recent Advances in Hematopoietic Stem Cell Gene Therapy

“…Although SIN vectors were developed from earlier retroviral [62] or lentiviral vectors [63] to reduce the risk of oncogenicity in SCID-X1 gene therapy, genotoxicity unrelated to mutations in gene insertion regions or γc chain gene overexpression have been reported with lentiviral vectors in recent years, and it seems that more sophisticated vector development is required [64].…”

Quantitative Shearing Linear Amplification Polymerase Chain Reaction: An Improved Method for Quantifying Lentiviral Vector Insertion Sites in Transplanted Hematopoietic Cell Systems

“…Vector copy number (VCN) was measured using quantitative PCR method. 26 One clone with an estimated VCN of 19 was chosen for further experiments. VCN was verified by Southern blot analysis and fluorescence in situ hybridization using a vector-specific probe.…”

“…We transduced Jurkat cells, a widely available human T cell line, with our CL20-4i-EF1a-hccOPT lentiviral vector 26,30 at high multiplicity of 100 and isolated single cell clones with defined copy numbers. As a standard, we chose one clone containing 19 copies of the vector genome per cell when measured by qPCR and Southern blot analysis for proviral DNA genome copies.…”

“…Jurkat cells were transduced with the CL20-4i-EF1a-hccOPT vector 26 at an MOI of 100 and were subsequently sorted into 96-well plates at 1 cell/well. Cell clones were expanded and genomic DNA was extracted from selected clones.…”

Eliminating SCID row: new approaches to SCID

“…38 A novel stable packaging cell line was derived at SJCRH and used to produce the clinical-grade vector. 39 Extensive preclinical safety studies were done that demonstrated undetectable genotoxocity from the vector, in contrast to the ␥-RVs with intact LTR used as a positive control.…”