X-linked severe combined immunodeficiency (SCID-X1) is a profound deficiency of T, B, and natural killer (NK) cell immunity caused by mutations in IL2RG encoding the common chain (γc) of several interleukin receptors. Gamma-retroviral (γRV) gene therapy of SCID-X1 infants without conditioning restores T cell immunity without B or NK cell correction, but similar treatment fails in older SCID-X1 children. We used a lentiviral gene therapy approach to treat five SCID-X1 patients with persistent immune dysfunction despite haploidentical hematopoietic stem cell (HSC) transplant in infancy. Follow-up data from two older patients demonstrate that lentiviral vector γc transduced autologous HSC gene therapy after nonmyeloablative busulfan conditioning achieves selective expansion of gene-marked T, NK, and B cells, which is associated with sustained restoration of humoral responses to immunization and clinical improvement at 2 to 3 years after treatment. Similar gene marking levels have been achieved in three younger patients, albeit with only 6 to 9 months of follow-up. Lentiviral gene therapy with reduced-intensity conditioning appears safe and can restore humoral immune function to posthaploidentical transplant older patients with SCID-X1.

IntroductionHIV-based lentiviral vectors are rapidly becoming the retrovirus vector system of choice for research and clinical gene transfer applications. The enhanced ability of lentiviral vectors to transduce both quiescent stem cells 1 and nondividing terminally differentiated cells 2 has led to the development of a wide range of therapeutic gene delivery vectors, 3 as well as promising research tools such as short hairpin RNA gene knockdown libraries 4 and vectors for induction of pluripotency in terminally differentiated cells. 5 Early gamma-retroviral clinical gene therapy vectors restored immune function in patients with X-linked severe combined immunodeficiency (SCID-X1), but they were subsequently found to cause proliferative disorders via transactivation of protooncogenes. 6,7 Newer lentiviral vector designs may significantly reduce that risk, and they await clinical testing for final validation of their predicted safety. Clinical-scale production of these vectors, however, is problematic, as the generation of stable producer cell lines is made significantly more difficult by their self-inactivating (SIN) long terminal repeats (LTRs). As a result, most clinical-grade production of lentiviral vectors is currently being performed using cumbersome transient transfection processes.Insertional mutagenesis by previous gamma-retroviral gene therapy vectors occurred when strong viral enhancers within the LTR activated genes (eg, LMO2) surrounding the integrated vector. 6,7 SIN vector designs completely eliminate the viral enhancers and promoters in the LTR, and when coupled with appropriate internal promoters having less or no enhancer activity, they have been shown to significantly reduce oncogene activation. [8][9][10] Chromatin insulator sequences have also been inserted into SIN LTRs and appear to protect neighboring genes from residual transactivation from the internal promoters. 11 When inserted into the LMO2 locus in Jurkat cells, lentiviral vector genomes containing an internal EF1␣ promoter flanked by SIN LTRs and chicken HS4 chromatin insulators caused only minimal transactivation of the LMO2 promoter. 12 Clinical-scale production of such safety-modified vectors would be greatly facilitated by stable producer cell lines, which allow convenient generation of standardized, large-volume supernatants for downstream process optimization and preclinical studies. Although there have been numerous reports of lentiviral packaging cell lines, 13-22 all high-titer (Ͼ 10 7 transducing units per milliliter [TU/mL]) stable producer lines described in these publications were created by the traditional method of viral transduction of packaging cell lines using non-SIN vector supernatants, which efficiently creates populations of cells with vector genomes integrated at sites favorable for active transcription, and in multiple copies per cell. SIN vector genomes, by virtue of the inactivating deletion in the LTR, are thus incompatible with this method. "Conditional SIN" vectors, 22 which contain regulatable enhancers ...

Lentiviral vectors are increasingly utilized in cell and gene therapy applications because they efficiently transduce target cells such as hematopoietic stem cells and T cells. Large-scale production of current Good Manufacturing Practices-grade lentiviral vectors is limited because of the adherent, serum-dependent nature of HEK293T cells used in the manufacturing process. To optimize large-scale clinical-grade lentiviral vector production, we developed an improved production scheme by adapting HEK293T cells to grow in suspension using commercially available and chemically defined serum-free media. Lentiviral vectors with titers equivalent to those of HEK293T cells were produced from SJ293TS cells using optimized transfection conditions that reduced the required amount of plasmid DNA by 50%. Furthermore, purification of SJ293TS-derived lentiviral vectors at 1 L yielded a recovery of 55% ± 14% (n = 138) of transducing units in the starting material, more than a 2-fold increase over historical yields from adherent HEK293T serum-dependent lentiviral vector preparations. SJ293TS cells were stable to produce lentiviral vectors over 4 months of continuous culture. SJ293TS-derived lentiviral vectors efficiently transduced primary hematopoietic stem cells and T cells from healthy donors. Overall, our SJ293TS cell line enables high-titer vector production in serum-free conditions while reducing the amount of input DNA required, resulting in a highly efficient manufacturing option.

Haemophilus ducreyi produces an outer membrane protein called DsrA, which is required for serum resistance. An isogenic dsrA mutant, FX517, was constructed previously in H. ducreyi 35000. Compared to its parent, FX517 cannot survive in normal human serum. When complemented in trans with a plasmid containing dsrA, FX517 is converted to a serum-resistant phenotype (C. Elkins, K. J. Morrow, Jr., and B. Olsen, Infect. Immun. 68:1608-1619, 2000). To test whether dsrA was transcribed in vivo, we successfully amplified transcripts in five biopsies obtained from four experimentally infected human subjects. To test whether DsrA was required for virulence, six volunteers were experimentally infected with 35000 and FX517 and observed for papule and pustule formation. Each subject was inoculated with two doses (70 to 80 CFU) of live 35000 and 1 dose of heat-killed bacteria on one arm and with three doses (ranging from 35 to 800 CFU) of live FX517 on the other arm. Papules developed at similar rates at sites inoculated with the mutant or parent. However, mutant papule surface areas were significantly smaller than parent papules. The pustule formation rate was 58% (95% confidence interval [CI] of 28 to 85%) at 12 parent sites, and 0% (95% CI of 0 to 15%) at 18 mutant sites (P ‫؍‬ 0.0004). Although biosafety regulations precluded our testing the complemented mutant in humans, these results suggest that expression of DsrA facilitates the ability of H. ducreyi to progress to the pustular stage of disease.