High-efficiency retroviral vector mediated gene transfer into human peripheral blood CD4+ T lymphocytes.

Genetic modification of peripheral blood T lymphocytes has been proposed as a therapeutic strategy for treating congenital disorders, cancer and viral diseases. Central to all T lymphocyte-based gene therapy strategies is the ability to efficiently and stably deliver genes into primary T lymphocytes. In this study, we sought to increase the gene transfer efficiency in CD4+ peripheral blood T lymphocytes using procedures which could be utilized in clinical applications. In order to quantity the gene transfer efficiency in primary CD4+ T cells, a high-titer retroviral vector which efficiently expresses a truncated version of the human low-affinity nerve growth factor receptor (delta LNGFR) was constructed. Transduced cells were then accurately enumerated with immunofluorescence staining and fluorescence activated cell sorting (FACS) analysis and rapidly isolated at high purity for further analysis. Using this system, a supernatant-based gene transfer procedure was developed which routinely yields gene transfer efficiencies of 25-40% into a wide repertoire of both freshly obtained and cryopreserved peripheral blood CD4+ T lymphocytes.

Efficient gene transfer with adeno-associated virus-based plasmids complexed to cationic liposomes for gene therapy of human prostate cancer.

We have shown previously that treatment of rats bearing the Dunning R3327 MatLyLu prostatic tumor with human interleukin 2 (IL-2) gene-modified tumor cell preparations induces potent antitumor immunity in the animal. To test the clinical feasibility of using genetically modified tumor vaccines for the treatment of prostate cancer, we have explored the use of a simplified gene delivery system based on liposomes to introduce and express the IL-2 gene in the Dunning rat R3327 MatLyLu prostatic tumor cell line (MatLyLu) and in short-term cultures of primary human prostatic tumor cells. Liposome-DNA complexes containing the adeno-associated virus inverted terminal repeats exhibited 3-10-fold higher levels of gene transfer and IL-2 expression than did liposome complexes with non-adeno-associated virus containing plasmids. Single transfections resulted in IL-2 expression for an extended period of time that exceeded severalfold the amount of IL-2 secreted from retrovirally transduced MatLyLu cells. X-irradiation of cells (4000 rads) prior to transfection did not affect cytokine secretion, indicating that liposome-mediated gene transfer does not depend on cell proliferation. High levels of gene transfer and IL-2 expression were also achieved in short-term cultures of primary human prostatic tumor cells established from tumor specimens obtained following radical prostatectomy of cancer patients. Depending on the type of liposome used, IL-2 levels secreted from the human prostatic tumor cells were comparable to or exceeded the levels of IL-2 secreted from retrovirally transduced MatLyLu cells, which induced antitumor immunity in the rat model. The ability to culture and expand ex vivo human prostatic tumor cells, and the use of a simple and highly efficient gene transfer method to generate genetically modified tumor vaccines, set the stage for clinical exploration of gene-based immunotherapy of prostate cancer.

Increased microvascular permeability contributes to preferential accumulation of Stealth liposomes in tumor tissue.

Stealth liposomes have recently emerged as a promising antitumor drug delivery system, yet no studies have been reported to examine their dynamic behavior at the microcirculatory level. In this investigation, we have used in vivo fluorescence videomicroscopy to study the decay in plasma concentration and the interstitial accumulation of Stealth and conventional liposomes in tumor and granulating tissue microcirculatory preparations. Fluorescently labeled Stealth or conventional liposomes were injected i.v. into rats bearing dorsal skinflap window chambers, some of which contained a vascularized mammary adenocarcinoma. After injection, fluorescent light intensities arising from liposomes within blood vessels and the interstitium were measured over time. These measurements were used to derive plasma pharmacokinetics and vascular permeability coefficients for each liposome species in both tumor and granulating normal tissues. Within the first 90 min after injection, Stealth liposome accumulation in the tumor interstitium was 3-4-fold that for conventional liposomes. The percentage of administered liposomes remaining in the circulation at the end of 90 min was 60.2% for Stealth and 20.4% for conventional liposomes. Tumor vascular permeability was 3.42 +/- 0.78 x 10(-7)cm/s for Stealth and 1.75 x 0.38 x 10(-7)cm/s for conventional liposomes. In normal granulating tissues permeability for the 2 constructs was equivalent at 0.8-0.9 x 10(-7)cm/s. In conclusion, preferential accumulation of Stealth liposomes in tumors was attributable to a combination of slower plasma clearance and higher vascular permeability relative to conventional liposomes. Our method of combining in vivo microscopy with a tumor microcirculatory model provides a unique approach to study quantitatively the delivery of liposomes to tumor tissues, since it can be used to study the process in real time at the microcirculatory level.

HIV-1 sensitivity to zidovudine and clinical outcome in children.

In adults with the acquired immunodeficiency syndrome, long-term monotherapy with zidovudine selects for human immunodeficiency virus type 1 (HIV-1) strains with substantially reduced in-vitro susceptibility to the drug. We have assessed the relation between in-vitro resistance to zidovudine and clinical outcome in children, in whom disease progression is more rapid than in adults. We studied 23 children with symptoms of HIV-1 disease during extended monotherapy with zidovudine. An in-vitro assay was used to determine the concentration of zidovudine required to inhibit by 50% the replication of viral isolates (IC50) obtained after 9 to 39 months of treatment. Viral stocks of high enough titre to yield reproducible results were obtained from 19 of the children. During the following 6 months of therapy, 9 children were stable, 7 deteriorated, and 3 died. There was a highly significant relation between decreased zidovudine susceptibility and poor clinical outcome (p less than 0.001) but no relation between IC50 and age at start of therapy or length of time on treatment. Age-adjusted CD4 lymphocyte counts were lower at the start of treatment (p = 0.02) and at the time of sampling (p = 0.01) in children whose viral isolates had an increased zidovudine IC50. Initial serum p24 antigen levels were not predictive of subsequent emergence of resistant virus, but at the time of sampling for viral sensitivity higher p24 antigen levels were associated with raised IC50 (p = 0.004). The findings suggest that most children who become unresponsive to monotherapy with zidovudine, as judged by clinical criteria, will have changes in in-vitro sensitivity to the drug. In these children, an alternative antiretroviral therapy should be considered.