RNA-based gene therapy for HIV with lentiviral vector-modified CD34(+) cells in patients undergoing transplantation for AIDS-related lymphoma

Abstract

AIDS patients who develop lymphoma are often treated with transplanted hematopoietic progenitor cells. As a first step in developing a hematopoietic cell-based gene therapy treatment, four patients undergoing treatment with these transplanted cells were also given gene-modified peripheral blood-derived (CD34(+)) hematopoietic progenitor cells expressing three RNA-based anti-HIV moieties (tat/rev short hairpin RNA, TAR decoy, and CCR5 ribozyme). In vitro analysis of these gene-modified cells showed no differences in their hematopoietic potential compared with nontransduced cells. In vitro estimates of successful expression of the anti-HIV moieties were initially as high as 22% but declined to approximately 1% over 4 weeks of culture. Ethical study design required that patients be transplanted with both gene-modified and unmanipulated hematopoietic progenitor cells obtained from the patient by apheresis. Transfected cells were successfully engrafted in all four infused patients by day 11, and there were no unexpected infusion-related toxicities. Persistent vector expression in multiple cell lineages was observed at low levels for up to 24 months, as was expression of the introduced small interfering RNA and ribozyme. Therefore, we have demonstrated stable vector expression in human blood cells after transplantation of autologous gene-modified hematopoietic progenitor cells. These results support the development of an RNA-based cell therapy platform for HIV.

Figure 1. Clinical Trial Design

Following Lymphoma Therapy, patients underwent hematopoietic progenitor cell mobilization with G-CSF as shown. The resulting apheresis product (HPC-A) was collected for up to 4 days; first to collect 2–5 ×10⁵ CD34+ cells/kg for the unmanipulated transplantation product (Fraction A) and then to collect CD34+ cells for the genetic modification (Fraction B). Unmanipulated HPC-A collections were cryopreserved immediately while HPC-A for genetic modification were enriched for CD34 content using a CliniMACS^™ device and then cryopreserved. The patients were treated with a myeloablative conditioning regimen (BCNU/VP16/Cytoxan as shown). Three days prior to transplantation (day -3) CD34-enriched HPC-A is thawed and transduced with the HIV-shI-TAR-CCR5Rz lentiviral vector. On day 0, the patients are infused with the gene modified HPC-A product followed by the unmanipulated HPC-A product on Day+1.

Figure 2. Genetic Marking of patient samples during in vitro culture

CD34-enriched HPC-A products were cultured in vitro following transduction for four weeks in liquid culture or on a murine stromal cell layer line in the presence of GM-CSF, IL3, IL6, Flt3L, erythropoietin, thrombopoietin. Cells were harvested weekly and DNA was extracted for qPCR analysis of the transgene. The average percentage of cells transduced with the HIV-shI-TAR-CCR5Rz genetic construct is defined as described in Methods. Copies of WPRE derived from a standard curve of known quantities of pHIV-shI- TAR-CCR5Rz plasmid spiked into the background of non transduced peripheral blood mononuclear cells. Apo B gene copies are determined from qPCR analysis of a standard curve of DNA from 10–1,000,000 peripheral blood mononuclear cells. (A) Average gene marking (bar) and individual values of gene marking (symbols) in bulk populations of cells from each patient over the 4 weeks of bulk liquid culture is shown. (B) For each patient, cells from in vitro culture were also sorted at 2 weeks (CD14, CD15, CD33, Gly-A and Lin-) or 4 weeks (CD10) for lineage specific marking analysis as described above (b).

Figure 3. RNA Expression in Pre-Infusion Transduced Cell Product

(A) Northern blot analysis of RNA expression in Patient 305 autologous CD34+ cells transduced with rHIV7-shI-TAR-CCR5RZ. Total RNA was extracted after transduction at day 13 and day 29. CEM cells were transduced with rHIV7-shI-TAR-CCR5RZ as a positive control. Non-transduced CD4+ cells were used as a negative control. 4 μg of total RNA was electrophoresed in an 8% polyacrylamide gel with 8 M urea, blotted onto a nylon membrane,and hybridized with a 32P-labeled probe for the siRNA, U16TAR decoy and anti-CCR5 ribozyme. (B) Quantitative PCR analysis of in vitro cultures for siRNA expression. Estimated siRNA copies per 8 ng of total cellular RNA is shown over time. Numbers of copies are estimated from a standard curve using known amounts of chemically synthesized tat/rev siRNA.

Figure 4. Gene Marking in the peripheral blood following HSC transplantation

DNA was isolated directly from peripheral blood samples obtained pre-apheresis and at 1–4, 6, 8, 10, 12, 18 and 24 months post infusion. Real-time PCR was performed to detect the presence of the Woodchuck post-transcriptional regulatory element (WPRE) sequence using primers specific for a 174 nt long fragment contained within the pHIV7-shITAR-CCR5RZ DNA construct and against a standard curve derived by titration of DNA isolated from a clone that contains a single-copy integration of the lentivirus into DNA isolated from PBMC. A parallel set of qPCR amplifications of all test and reference samples using primers specific for the human p21 promoter was used to normalize input DNA for the WPRE amplifications. Sample values were considered quantifiable if they were at or above the limit of quantification (LOQ), defined as the lowest dilution of the H9c1 DNA standard curve for which at least 2 of the 3 replicates provide a measurable Ct value with a single expected melting curve. They were considered detectable if they were below the LOQ but above the limit of detection (LOD) defined as the lowest sample concentration where at least 1 of the 3 replicates generated a detectable Ct value with a single expected melting curve. LOQ and LOD values were determined for each amplification reaction and typically were in the range of 0.05% (500 cells/million) and 0.01% (100 cells/million), respectively. Open symbols on the Y axis (0 timepoint) indicate level of gene marking estimated for each patient as shown in Table 1.