Vectors derived from human immunodeficiency virus (HIV) are highly efficient vehicles for in vivo gene delivery. However, their biosafety is of major concern. Here we exploit the complexity of the HIV genome to provide lentivirus vectors with novel biosafety features. In addition to the structural genes, HIV contains two regulatory genes, tat and rev, that are essential for HIV replication, and four accessory genes that encode critical virulence factors. We previously reported that the HIV type 1 accessory open reading frames are dispensable for efficient gene transduction by a lentivirus vector. We now demonstrate that the requirement for the tat gene can be offset by placing constitutive promoters upstream of the vector transcript. Vectors generated from constructs containing such a chimeric long terminal repeat (LTR) transduced neurons in vivo at very high efficiency, whether or not they were produced in the presence of Tat. When the rev gene was also deleted from the packaging construct, expression of gag and pol was strictly dependent on Rev complementation in trans. By the combined use of a separate nonoverlapping Rev expression plasmid and a 5′ LTR chimeric transfer construct, we achieved optimal yields of vector of high transducing efficiency (up to 107transducing units [TU]/ml and 104 TU/ng of p24). This third-generation lentivirus vector uses only a fractional set of HIV genes: gag, pol, and rev. Moreover, the HIV-derived constructs, and any recombinant between them, are contingent on upstream elements and trans complementation for expression and thus are nonfunctional outside of the vector producer cells. This split-genome, conditional packaging system is based on existing viral sequences and acts as a built-in device against the generation of productive recombinants. While the actual biosafety of the vector will ultimately be proven in vivo, the improved design presented here should facilitate testing of lentivirus vectors.

High-grade epithelial ovarian carcinomas containing mutated BRCA1 or BRCA2 (BRCA1/2) homologous recombination (HR) genes are sensitive to platinum-based chemotherapy and PARP inhibitors (PARPi), while restoration of HR function due to secondary mutations in BRCA1/2 has been recognized as an important resistance mechanism. We sequenced core HR pathway genes in 12 pairs of pretreatment and postprogression tumor biopsy samples collected from patients in ARIEL2 Part 1, a phase II study of the PARPi rucaparib as treatment for platinum-sensitive, relapsed ovarian carcinoma. In 6 of 12 pretreatment biopsies, a truncation mutation in BRCA1, RAD51C, or RAD51D was identified. In five of six paired postprogression biopsies, one or more secondary mutations restored the open reading frame. Four distinct secondary mutations and spatial heterogeneity were observed for RAD51C. In vitro complementation assays and a patient-derived xenograft, as well as predictive molecular modeling, confirmed that resistance to rucaparib was associated with secondary mutations. Significance Analyses of primary and secondary mutations in RAD51C and RAD51D provide evidence for these primary mutations in conferring PARPi sensitivity and secondary mutations as a mechanism of acquired PARPi resistance. PARPi resistance due to secondary mutations underpins the need for early delivery of PARPi therapy and for combination strategies.

Purpose: This trial evaluated the safety, clinical activity, and immunogenicity of an allogeneic cellular immunotherapy in 55 chemotherapy-naI« ve patients with hormone-refractory prostate cancer (HRPC). The immunotherapy, based on the GVAX platform, is a combination of two prostate carcinoma cell lines modified with the granulocyte macrophage colony-stimulating factor (GM-CSF) gene. Experimental Design: HRPC patients with radiologic metastases (n = 34) or rising prostatespecific antigen (PSA) only (n = 21) received a prime dose of 500 million cells and 12 boost doses of either100 million cells (low dose) or 300 million cells (high dose) biweekly for 6 months. End points were changes in PSA, time to progression, and survival. Results: Median survival was 26.2 months (95% confidence interval, 17, 36) in the radiologic group: 34.9 months (8, 57) after treatment with the high dose (n = 10) of immunotherapy and 24.0 months (11, 35) with the low dose (n = 24).The median time to bone scan progression in the radiologic group was 5.0 months (2.6, 11.6) with the high dose and 2.8 months (2.8, 5.7) with the low dose. In the rising-PSA group (n = 21) receiving the low dose, the median time to bone scan progression was 5.9 months (5.6, not reached), and median survival was 37.5 months (29, 56). No dose-limiting or autoimmune toxicities were seen; the most common adverse events were injection site reaction and fatigue. Conclusions: These results suggest that this GM-CSF^secreting, allogeneic cellular immunotherapy is well tolerated and may have clinical activity in patients with metastatic HRPC. Phase 3 trials to confirm these results are under way.Approximately 27,050 men die annually from metastatic hormone-refractory prostate cancer (HRPC; ref. 1). Although chemotherapy with docetaxel has been shown to prolong survival in HRPC (2, 3), alternatives to chemotherapy remain of considerable interest to many patients and physicians. Recent advances in the understanding of cancer immunology have led to the development of new cancer treatments specifically designed to stimulate the patient's immune system. Although prostate cancer has traditionally been thought of as poorly immunogenic, numerous studies have shown that tumor tolerance can be reversed (4 -6). Prostate cancer is a good target for immunotherapy due to the typically slow growth rate of most prostate tumor cells, which in turn permits an appropriately stimulated immune system time to mount antitumor responses (4, 5).Immunotherapy typically involves presenting one or more tumor antigens to the patient's immune system in vivo or to harvested immune cells in vitro (4, 6). An immune system stimulant may be included in the treatment to enhance the immune response to the antigens. Whole tumor cells have been proposed as an antigen source in immunotherapy because relevant prostate cancer tumor-rejection antigens have not been convincingly identified, and a polyvalent source of antigens can better address ''antigen escape'' resulting from the modulation and down-reg...

The plasmid-borne mph(A) gene that confers resistance to azithromycin and has recently emerged in Shigella sonnei is present in multidrug- and non–multidrug-resistant Escherichia coli isolates from 4 continents. Further spread of mph(A) to Shigella and Salmonella spp. may be expected.

Purpose Fibroblast activation protein (FAP) is a membrane-bound protease that has limited expression in normal adult tissues but is highly expressed in the tumor microenvironment of many solid cancers. FAP-2286 is a FAP-binding peptide coupled to a radionuclide chelator that is currently being investigated in patients as an imaging and therapeutic agent. The potency, selectivity, and efficacy of FAP-2286 were evaluated in preclinical studies. Methods FAP expression analysis was performed by immunohistochemistry and autoradiography on primary human cancer specimens. FAP-2286 was assessed in biochemical and cellular assays and in in vivo imaging and efficacy studies, and was further evaluated against FAPI-46, a small molecule–based FAP-targeting agent. Results Immunohistochemistry confirmed elevated levels of FAP expression in multiple tumor types including pancreatic, breast, and sarcoma, which correlated with FAP binding by FAP-2286 autoradiography. FAP-2286 and its metal complexes demonstrated high affinity to FAP recombinant protein and cell surface FAP expressed on fibroblasts. Biodistribution studies in mice showed rapid and persistent uptake of 68Ga-FAP-2286, 111In-FAP-2286, and 177Lu-FAP-2286 in FAP-positive tumors, with renal clearance and minimal uptake in normal tissues. 177Lu-FAP-2286 exhibited antitumor activity in FAP-expressing HEK293 tumors and sarcoma patient-derived xenografts, with no significant weight loss. In addition, FAP-2286 maintained longer tumor retention and suppression in comparison to FAPI-46. Conclusion In preclinical models, radiolabeled FAP-2286 demonstrated high tumor uptake and retention, as well as potent efficacy in FAP-positive tumors. These results support clinical development of 68Ga-FAP-2286 for imaging and 177Lu-FAP-2286 for therapeutic use in a broad spectrum of FAP-positive tumors.