AIDS patients undergoing autologous transplantation for lymphoma were treated with gene-modified peripheral blood derived (CD34+) hematopoietic progenitor cells (HPC) expressing 3 RNA-based anti-HIV moieties (Tat/Rev shRNA, TAR decoy and CCR5 ribozyme). In vitro analysis of gene-modified HPC showed no differences in the hematopoietic potential compared with non-transduced cells. In vitro estimates of gene marking were as high as 22% but declined to ~1% over 4 weeks of culture. Ethical study design required that patients were transplanted with both gene modified and unmanipulated hematopoietic progenitor cell apheresis products (HPC-A). All 4 infused patients engrafted (ANC>500) by day 11 post-infusion and showed no unexpected infusion related toxicities. Persistent vector marking in multiple cell lineages has been observed at low levels for up to 24 months as has expression of siRNA and ribozyme. This is the first demonstration of siRNA expression in human blood cells following transplantation of autologous gene-modified CD34+ HPC. These results support the development of an RNA-based cell therapy platform for HIV. Summary Stem cell gene therapy for HIV results in sustained RNA expression in the blood of patients for up to 2 years following transplant.

Vectors derived from human immunodeficiency virus (HIV) hold promise for efficient gene delivery into human hematopoietic cells. In this study, HIV vectors containing different combinations of cis-acting elements, including the HIV central flap sequence, and the woodchuck posttranscriptional regulatory element (WPRE) in combination with two different promoters, were used to transduce primary human lymphocytes and cord blood CD34+ progenitor cells. The effect of these elements on the transduction efficiency and transgene expression was systematically evaluated. The results demonstrate that with the combination of flap, WPRE sequences, and the promoter derived from spleen focus-forming virus (SFFV), a foreign gene can be efficiently delivered into primary human T lymphocytes and cord blood CD34+ cells. The study establishes the parameters for proper vector design to efficiently deliver foreign genes into human hematopoietic cells.

Gene therapeutic strategies show promise in controlling human immunodeficiency virus (HIV) infection and in restoring immunological function. A number of efficacious anti-HIV gene constructs have been described so far, including small interfering RNAs (siRNAs), RNA decoys, transdominant proteins, and ribozymes, each with a different mode of action. However, as HIV is prone to generating escape mutants, the use of a single anti-HIV construct would not be adequate to afford long range-viral protection. On this basis, a combination of highly potent anti-HIV genes--namely, a short hairpin siRNA (shRNA) targeting rev and tat, a transactivation response (TAR) decoy, and a CCR5 ribozyme--have been inserted into a third-generation lentiviral vector. Our recent in vitro studies with this construct, Triple-R, established its efficacy in both T-cell lines and CD34 cell-derived macrophages. In this study, we have evaluated this combinatorial vector in vivo. Vector-transduced CD34 cells were injected into severe combined immunodeficiency (SCID)-hu mouse thy/liv grafts to determine their capacity to give rise to T cells. Our results show that phenotypically normal transgenic T cells are generated that are able to resist HIV-1 infection when challenged in vitro. These important attributes of this combinatorial vector show its promise as an excellent candidate for use in human clinical trials.

A patient without evident immune deficiency who received a transfusion of blood from a second-degree family member developed fatal transfusion-associated graft-versus-host disease (TA-GVHD). The donor was homozygous for an HLA haplotype for which the recipient was heterozygous (one-way HLA match). All 39 reported cases of TA-GVHD in immunocompetent patients were reviewed to ascertain the predisposing factors and to define the indications for irradiating blood for this population. HLA typing was described in 15 cases; in 13, including seven related and six unrelated donors, a one-way HLA match was present. Thirty-one (79%) of the 39 cases were reported from Japan (and 196 other cases are cited in the Japanese literature), but a one-way HLA match among unrelated donors at HLA-A, -B, -DR loci is only approximately two to four times more likely in Japanese persons than in whites. Fresh blood (< 96 hours old) was used in 29 (94%) of the 31 cases reported from Japan and in 33 (87%) of 38 cases overall (in one case, the age of the blood used was not reported). Thus, factors that appear to predispose to TA-GVHD in immunocompetent patients are a one-way HLA match, fresh blood, and, possibly, Japanese ancestry. Irradiating cellular blood components from all blood relatives of transfusion recipients will not completely eliminate the risk of TA-GVHD.