Intraprostatic distribution and long-term follow-up after AdV-tk immunotherapy as neoadjuvant to surgery in patients with prostate cancer.

A phase I-II study to evaluate gene-mediated cytotoxic immunotherapy in newly diagnosed prostate cancer before radical prostatectomy was conducted in Monterrey, Mexico. First, to investigate delivery of adenovirus to the prostate, fluorescently labeled vector was injected into fresh prostatectomy specimens and distribution was visually analyzed. The optimal volume and site instillation was then used for transrectal ultrasound guided intraprostatic injection in 10 patients with adenocarcinoma scheduled for radical prostatectomy. Each received two apical and two basal 0.5 ml injections of AdV-tk for a total of 1 × 10(11) vp followed by 14 days of prodrug. Nine patients continued to tumor resection: six high risk, one intermediate and two low risk. In vivo vector distribution was analyzed from the resected tissue of four patients. Patients were monitored for tumor progression and acute and long-term safety. For vector delivery, two apical and two basal injections of 0.5 ml led to optimal organ-wide distribution ex vivo and in vivo. Cytotoxicity was evidenced by transient rise in PSA and tumor histology. There were no significant adverse events deemed related to the treatment and no late toxicities after median follow-up of 11.3 years. All six high-risk patients had positive surgical margins and one had seminal vesicle involvement. Despite slow PSA rise post surgery in three of these patients, none developed metastases. The intermediate- and low-risk patients had complete resections and none have progressed. In conclusion, in vivo transrectal ultrasound guided instillation of an adenoviral vector into four sites in the prostate was practical as an outpatient procedure, well tolerated and led to distribution throughout the intraprostatic tumor mass. AdV-tk demonstrated no significant acute or late toxicities. Trends in PSA and disease progression conveyed the possibility of a sustained immune response against residual disease.

Cirrhotic rat livers with extensive fibrosis can be safely transduced with clinical-grade adenoviral vectors. Evidence of cirrhosis reversion.

Adenoviral vectors efficiently target normal liver cells; however, a clear-cut description of the safety boundaries for using adenovectors in hepatic cirrhosis has not been settled. With this in mind, we used a first-generation, replication-deficient adenoviral vector carrying the E. coli lacZ gene (Ad5betaGal) to monitor therapeutic range, biodistribution, toxicity and transduction efficiency in Wistar rats made cirrhotic by two different experimental approaches resembling alcoholic cirrhosis and biliary cirrhosis in humans. Further, we show proof of concept on fibrosis reversion by a 'therapeutic' Ad-vector (AdMMP8) carrying a gene coding for a collagen-degrading enzyme. Dose-response experiments with Ad5betaGal ranging from 1 x 10(8)-3 x 10(12) viral particles (vp) per rat (250 g), demonstrated that adenovirus-mediated gene transfer via iliac vein at 3 x 10(11 )vp/rat, resulted in an approximately 40% transduction in livers of rats made cirrhotic by chronic intoxication with carbon tetrachloride, compared with approximately 80% in control non-cirrhotic livers. In rats made cirrhotic by bile-duct obstruction only, 10% efficiency of transduction was observed. Biodistribution analyses showed that vector expression was detected primarily in liver and at a low level in spleen and kidney. Although there was an important increase in liver enzymes between the first 48 h after adenovirus injection in cirrhotic animals compared to non-transduced cirrhotic rats, this hepatic damage was resolved after 72-96 h. Then, the cDNA for neutrophil collagenase, also known as Matrix Metalloproteinase 8 (MMP8), was cloned in an Ad-vector and delivered to cirrhotic rat livers being able to reverse fibrosis in 44%. This study demonstrates the potential use of adenoviral vectors in safe transient gene therapy strategies for human liver cirrhosis.

In vivo surgical resection plus adjuvant gene therapy in the treatment of mammary and prostate cancer.

Adenoviral-mediated gene therapy delivery, combining the herpes simplex virus thymidine kinase gene (Ad-tk) with gancyclovir, has been evaluated as a treatment modality for numerous tumors in the laboratory and in the clinics. As a single modality, gene therapy has shown some promising local and systemic results but no curative success. Surgery is the standard of care for many solid tumors. However, minor residual tumor following surgical resection can lead to local recurrence, and surgery is neither efficient nor plausible for metastatic disease. In this study, two tumor models were used to evaluate the effects of Ad-tk gene therapy as an adjuvant to surgery. Subcutaneous mammary- and prostate-derived tumors were produced in syngeneic mice. To evaluate systemic effects, tumor cells were injected intravenously, with subsequent formation of lung nodules. The subcutaneous tumors were surgically resected and the tumor bed was bathed with saline or Ad-tk. The animals were evaluated for toxicity, local tumor recurrence, survival, and lung nodule formation. No evidence of additional toxicity was observed. In the less aggressive mammary model, the time to recurrence was increased from 11.7 (+/-1.0) days to 22.7 (+/-5.5) days. In the prostate model, recurrence went from a mean of 17.3 (+/-5.6) to 22.6 (+/-6.8) days. Survival was also improved from a mean of 19.7 (+/-1.1) to 32.3 (+/-4.8) and 26.1 (+/-5.0) to 34.1 (+/-6.1) days in the mammary and prostate models, respectively. Evidence of systemic benefits from the use of adjuvant Ad-tk therapy was demonstrated by a significant reduction in lung nodules from a mean of 17 to 3.5. These results suggest that Ad-tk gene therapy may be a useful adjuvant for patients undergoing surgery for treatment of cancer.

Enhanced therapeutic effect of HSV-tk+GCV gene therapy and ionizing radiation for prostate cancer.

Standard therapies for prostate cancer including radiation, prostatectomy, and hormone ablation have significant toxicities and recurrence risk. HSV-tk gene therapy may be effective in combination with radiation therapy due to complementary mechanisms and distinct toxicity profiles. Mouse prostate tumors transplanted subcutaneously were treated by either gene therapy involving intratumoral injection of AdV-tk followed by systemic ganciclovir or local radiation therapy or the combination of gene and radiation therapy. Both single-therapy modalities showed a 38% decrease in tumor growth compared to controls. The combined treatment resulted in a decrease of 61%. In addition the combined-therapy group had a mean survival of 22 days versus 16.6 days for single therapy and 13.8 days for nontreated controls. To analyze systemic anti-tumor activity, lung metastases were generated by tail vein injection of RM-1 prostate cancer cells on the same day that they were injected subcutaneously. The primary tumors were treated as before with AdV-tk followed by ganciclovir, radiation, or the combination. The number of lung nodules was reduced by 37% following treatment with AdV-tk, whereas radiotherapy alone had no effect on metastatic growth. The combination led to an additional 50% reduction in lung colonization. Primary tumors that received the combination therapy had a marked increase in CD4 T cell infiltrate. This is the first report showing a dramatic systemic effect following the local combination treatment of radiation and AdV-tk. A clinical study using this strategy has been initiated and patient accrual is ongoing.

Liver cirrhosis is reverted by urokinase-type plasminogen activator gene therapy.

Liver cirrhosis represents a worldwide health problem and is a major cause of mortality. Cirrhosis is the result of extensive hepatocyte death and fibrosis induced by chronic alcohol abuse and hepatitis B and C viruses. Successful gene therapy approaches to this disease may require both reversal of fibrosis and stimulation of hepatocyte growth. Urokinase-type plasminogen activator (uPA) may serve this function, as it is an initiator of the matrix proteolysis cascade and induces hepatocyte growth factor expression. In a rat cirrhosis model, a single iv administration of a replication-deficient adenoviral vector encoding a nonsecreted form of human uPA resulted in high production of functional uPA protein in the liver. This led to induction of collagenase expression and reversal of fibrosis with concomitant hepatocyte and improved liver function. Thus, uPA gene therapy may be an effective strategy for treating cirrhosis in humans.

Reporting adverse events in gene therapy studies.

The reporting of adverse events occurring in gene therapy studies is currently under discussion. There are many agencies involved in the reporting of adverse events, often with reporting guidelines that are unique to each organisation. Guidelines differ according to the type of event (adverse event, adverse drug reaction, unexpected adverse drug reaction, serious adverse event and serious adverse drug reaction). While there is a move toward making guidelines more stringent in the US, there is concern that this will increase the number of reports of unrelated events, create confusion from a global perspective, and increase public concern needlessly.

Lymphoproliferative disorders involving Epstein-Barr virus after hemopoietic stem cell transplantation.

Lymphoproliferative disorders involving uncontrolled expansion of donor-derived B cells infected with Epstein-Barr virus (EBV) are a significant problem after hemopoietic stem cell transplantation. Risk factors, which include T cell depletion, major histocompatibility complex mismatch, and intensity of immunosuppression illustrate the importance of T cell immune surveillance. Recent studies have identified viral and host factors that affect the T-cell response to EBV. Monitoring EBV load in the blood by polymerase chain reaction allows early identification of high-risk patients and early institution of therapy. Adoptive immunotherapy approaches using donor T cells have proven effective and EBV-specific cytotoxic T lymphocytes have also been used successfully for prophylaxis. The simplest way of preventing EBV lymphoproliferation, however, may be to deplete B cells from the donor marrow prior to infusion to prevent the transmission of EBV-infected B cells.

Thymic nurse cells are sites of thymocyte apoptosis.

Thymic stromal cells play important roles in thymocyte differentiation and selection. Thymic nurse cells (TNC) are thymic epithelial stromal cells that envelop two to 200 CD4+CD8+ thymocytes (TNC-T). The mechanism by which TNC complexes form and their role in thymocyte development are unknown. TNC have been implicated as specialized microenvironments for proliferation, positive selection, negative selection, and apoptosis. Using TCR-gamma junctional sequence analysis of thymocytes within individual TNC, eight of ten TNC analyzed were polyclonal, and two showed evidence of oligoclonality. TCR-alpha beta expression was not detectable on most TNC-T and SCID mice, which do not express TCRs because of a defect in TCR gene rearrangement, had normal numbers of thymocyte-bearing TNC. Thus, TCR expression is not necessary for TNC formation. Treatment of mice with Abs to CD3 epsilon, which induces apoptosis in immature thymocytes, resulted in an eightfold increase in TNC per thymus with 95.5% apoptotic TNC-T. These results suggest that a function of TNC is the clearance of nonfunctional, nonselected, apoptotic thymocytes.

V gamma 3 T cell receptor rearrangement and expression in the adult thymus.

Rearrangement and expression of the V gamma 3-J gamma 1 TCR has been found in murine dendritic epidermal cells (DEC) and fetal thymus. By using the polymerase chain reaction technique, V gamma 3-J gamma 1 rearrangements and RNA expression were detected in the murine adult thymus. Individual genomic and cDNA junctions were cloned and sequenced. In genomic DNA, 55% (16/29) of V gamma 3-J gamma 1 junctional sequences had N regions ranging in length from 1 to 12 nucleotides resulting in considerable junctional diversity. Only 5% (2/42) of cDNA sequences had N regions. The canonical DEC sequence represented 36% (15/42) of the cDNA sequences. Thus, fetal-type V gamma 3-J gamma 1 rearrangements lacking N regions were preferentially expressed in adult thymocytes, some of which may be DEC precursors. The developmental stages in which V gamma 3-J gamma 1 rearrangements are generated were studied by using polymerase chain reaction to detect circular rearrangement products. Active V gamma 3-J gamma 1 rearrangement was detected in thymuses from fetal, newborn, and 2-wk-old mice but not in 5-wk or 8-wk-old (adult) mice. V gamma 2, one of the most common V gamma rearrangements in the adult, was found to be actively rearranging to J gamma 1 in the adult thymus. However, V gamma 2-V gamma 3 replacement rearrangement was not found. These results support the hypotheses that adult thymocytes with rearranged V gamma 3-J gamma 1 are persistent from earlier developmental stages and represent a separate lineage from those with V gamma 2-J gamma 1 rearrangements.