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.

The HIV-1 coreceptor CCR5 is a validated target for HIV/AIDS therapy. The apparent elimination of HIV-1 in a patient treated with an allogeneic stem cell transplant homozygous for a naturally occurring CCR5 deletion mutation (CCR5Δ32/Δ32) supports the concept that a single dose of HIV-resistant hematopoietic stem cells can provide disease protection. Given the low frequency of naturally occurring CCR5Δ32/Δ32 donors, we reasoned that engineered autologous CD34+ hematopoietic stem/progenitor cells (HSPCs) could be used for AIDS therapy. We evaluated disruption of CCR5 gene expression in HSPCs isolated from granulocyte colony-stimulating factor (CSF)-mobilized adult blood using a recombinant adenoviral vector encoding a CCR5-specific pair of zinc finger nucleases (CCR5-ZFN). Our results demonstrate that CCR5-ZFN RNA and protein expression from the adenoviral vector is enhanced by pretreatment of HSPC with protein kinase C (PKC) activators resulting in >25% CCR5 gene disruption and that activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway is responsible for this activity. Importantly, using an optimized dose of PKC activator and adenoviral vector we could generate CCR5-modified HSPCs which engraft in a humanized mouse model (albeit at a reduced level) and support multilineage differentiation in vitro and in vivo. Together, these data establish the basis for improved approaches exploiting adenoviral vector delivery in the modification of HSPCs.

The 78-kDa glucose-regulated protein (GRP78) is a major endoplasmic reticulum (ER) protein that can form stable associations with a variety of proteins retained in the ER because of underglycosylation or other conformational changes. In this study, we provide evidence at the transcriptional level that a conformationally abnormal protein, an altered herpes simplex virus type 1 envelope protein that is retained in the ER of a mammalian cell line, transactivates the grp78 promoter. In contrast, the normal viral envelope glycoprotein does not elevate grp78 promoter activity. Using a series of 5' deletions, linker-scanning, and internal deletion mutations spanning a 100-bp region from -179 to -80, we correlate the cis-acting regulatory elements mediating the activation of grp78 by malfolded proteins, glycosylation block, and the calcium ionophore A23187. We show that they all act through the same control elements, suggesting that they share a common signal. We report here that the highly conserved grp element, while important for basal level and induced grp78 expression, is functionally redundant. The single most important element, by linker-scanning analysis, is a 10-bp region that contains a CCAAT motif. It alone is not sufficient for promoter activity, but a 40-bp region (-129 to -90) that contains this motif is essential for mediating basal level and stress inducibility of the grp78 promoter. We show that the transcription factor CTF/NF-I is able to transactivate the grp78 promoter through interaction with this CCAAT motif.

GRP78, a 78,000 dalton protein residing in the endoplasmic reticulum, is postulated to play important roles in protein folding and cell survival during calcium and other physiological stress. Here we describe the construction of an eukaryotic expression vector for the constitutive expression of grp78 antisense RNA and the creation of a CHO cell line, 78WO, which expresses high levels of the grp78 antisense RNA through amplification of the stably transfected antisense vector. We observed that whereas 78WO maintains a basal level of GRP78 similar to that of control cells, GRP78 is no longer inducible by A23187. The 78WO cells have undergone a compensatory increase in grp78 transcription such that the effects of antisense are cancelled out at the protein level under nonstressed conditions. In these same cells, GRP94, a 94,00 dalton ER protein, is also rendered noninducible by A23187. This provides the first evidence that the regulation of two ER proteins might be coupled such that the failure to induce GRP78 results in the down-regulation of GRP94. The 78WO cell line grows with a doubling time of about 26 hr and exhibits decreased tolerance to A23187, suggesting the GRPs contribute to cell viability under calcium stress. The establishment of this cell line, which can be stably maintained, will provide a useful tool for testing whether the induction of the GRPs is important for protein folding or transport and whether their enhanced synthesis is the cause or consequence of a variety of physiological adaptations.