A phase I clinical trial of oncolytic adenovirus mediated suicide and interleukin-12 gene therapy in patients with recurrent localized prostate adenocarcinoma.

In a phase I dose escalation and safety study (NCT02555397), a replication-competent oncolytic adenovirus expressing yCD, TK and hIL-12 (Ad5-yCD/mutTKSR39rep-hIL-12) was administered in 15 subjects with localized recurrent prostate cancer (T1c-T2) at increasing doses (1 × 1010, to 1 × 1012 viral particles) followed by 7-day treatment of 5-fluorocytosine (5-FC) and valganciclovir (vGCV). The primary endpoint was toxicity through day 30 while the secondary and exploratory endpoints were quantitation of IL-12, IFNγ, CXCL10 and peripheral blood mononuclear cells (PBMC). The study maximum tolerated dose (MTD) was not reached indicating 1012 viral particles was safe. Total 115 adverse events were observed, most of which (92%) were grade 1/2 that did not require any treatment. Adenoviral DNA was detected only in two patients. Increase in IL-12, IFNγ, and CXCL10 was observed in 57%, 93%, and 79% patients, respectively. Serum cytokines demonstrated viral dose dependency, especially apparent in the highest-dose cohorts. PBMC analysis revealed immune system activation after gene therapy in cohort 5. The PSA doubling time (PSADT) pre and post treatment has a median of 1.55 years vs 1.18 years. This trial confirmed that replication-competent Ad5-IL-12 adenovirus (Ad5-yCD/mutTKSR39rep-hIL-12) was well tolerated when administered locally to prostate tumors.

Phase I trial of oncolytic adenovirus-mediated cytotoxic and interleukin-12 gene therapy for the treatment of metastatic pancreatic cancer.

The safety of oncolytic adenovirus-mediated suicide and interleukin-12 (IL 12) gene therapy was evaluated in metastatic pancreatic cancer patients. In this phase I study, a replication-competent adenovirus (Ad5-yCD/mutTKSR39 rep-hIL-12) expressing yCD/mutTKSR39 (yeast cytidine deaminase/mutant S39R HSV-1 thymidine kinase) and human IL-12 (IL 12) was injected into tumors of 12 subjects with metastatic pancreatic cancer (T2N0M1-T4N1M1) at escalating doses (1 × 1011, 3 × 1011, or 1 × 1012 viral particles). Subjects received 5-fluorocytosine (5-FC) therapy for 7 days followed by chemotherapy (FOLFIRINOX or gemcitabine/albumin-bound paclitaxel) starting 21 days after adenovirus injection. The study endpoint was toxicity through day 21. Experimental endpoints included measurements of serum IL 12, interferon gamma (IFNG), and CXCL10 to assess immune system activation. Peripheral blood mononuclear cells and proliferation markers were analyzed by flow cytometry. Twelve patients received Ad5-yCD/mutTKSR39 rep-hIL-12 and oral 5-FC. Approximately 94% of the 121 adverse events observed were grade 1/2 requiring no medical intervention. Ad5-yCD/mutTKSR39 rep-hIL-12 DNA was detected in the blood of two patients. Elevated serum IL 12, IFNG, and CXCL10 levels were detected in 42%, 75%, and 92% of subjects, respectively. Analysis of immune cell populations indicated activation after Ad5-yCD/mutTKSR39 rep-hIL-12 administration. The median survival of patients in the third cohort is 18.1 (range, 3.5-20.0) months. The study maximum tolerated dose (MTD) was not reached.

Do Sociodemographic Factors Influence Outcome in Prostate Cancer Patients Treated With External Beam Radiation Therapy?

OBJECTIVES: The purpose of this study was to analyze the prognostic significance of sociodemographic factors on biochemical control (bNED) and overall survival (OS) in patients with prostate cancer. METHODS: Prostate cancer patients treated with definitive external beam radiation therapy (EBRT)±hormone therapy from 1997 to 2006 were analyzed in this IRB-approved study. Patient demographics, treatment (Tx), and clinical outcome were obtained from electronic medical records. Median household income (mHHI) at the census block group level was obtained from the 2000 census data. Data on disease and Tx parameters included Gleason score, pre-Tx prostate-specific antigen (PSA), T stage, year of Tx, EBRT dose, and use of hormone therapy. Patients were categorized as having low-risk, intermediate-risk, or high-risk disease. Sociodemographic factors included age, race, marital status, and mHHI. Biochemical failure was defined as nadir PSA+2 ng/mL. OS was based on death from any cause. RESULTS: A total of 788 consecutive patients were studied with a median follow-up of 7 years (range, 0.4 to 15 y). African Americans comprised 48% of the patients, whereas 46% of patients were white and 6% were other races. Whites had an average mHHI of $60,190 compared with $36,917 for African Americans (P<0.001). After multivariable modeling, only radiation dose was predictive for bNED (P=0.004) or OS (P=0.008). No sociodemographic factors were predictive for either outcome. Higher radiation dose predicted for better biochemical control and OS. CONCLUSIONS: This analysis suggests that sociodemographic factors are not important prognostic factors in determining outcome after EBRT for prostate cancer.

Preclinical toxicology of oncolytic adenovirus-mediated cytotoxic and interleukin-12 gene therapy for prostate cancer.

The purpose of this study was to examine the toxicity of combining oncolytic adenovirus-mediated cytotoxic and interleukin 12 (IL-12) gene therapy in a preclinical model to support future phase 1 trials. One hundred and twenty C57BL/6 male mice received an intraprostatic injection of saline (n = 24) or an oncolytic adenovirus (Ad5-yCD/mutTKSR39rep-mIL12) expressing two suicide genes and mouse IL-12 (n = 96). The adenovirus was administered at three dose levels (1.3 × 106, 1.3 × 107, 1.3 × 108 vp/kg) followed by 2 weeks of 5-flurocytosine (5-FC) and gancliclovir (GCV) prodrug therapy. There were no premature deaths. Daily observations of animals did not reveal any obvious clinical problems throughout the 78-day in-life phase of the study. Animals in the highest adenovirus dose group exhibited lymphopenia and transaminitis on day 3, both of which resolved by day 17. Except for mild inflammation of the prostate and seminal vesicles, histopathology of major organs was largely unremarkable. IL-12 and interferon-gamma levels in prostate and serum peaked on day 3 and were either undetectable or returned to baseline levels by day 17. No adenoviral DNA was detected in serum in any group at any time point. The results demonstrate that local administration of an oncolytic adenovirus expressing two suicide genes and IL-12 is well tolerated and support moving this investigational approach into human trials.

Prospective randomized phase 2 trial of intensity modulated radiation therapy with or without oncolytic adenovirus-mediated cytotoxic gene therapy in intermediate-risk prostate cancer.

PURPOSE: To assess the safety and efficacy of combining oncolytic adenovirus-mediated cytotoxic gene therapy (OAMCGT) with intensity modulated radiation therapy (IMRT) in intermediate-risk prostate cancer. METHODS AND MATERIALS: Forty-four men with intermediate-risk prostate cancer were randomly assigned to receive either OAMCGT plus IMRT (arm 1; n=21) or IMRT only (arm 2; n=23). The primary phase 2 endpoint was acute (≤90 days) toxicity. Secondary endpoints included quality of life (QOL), prostate biopsy (12-core) positivity at 2 years, freedom from biochemical/clinical failure (FFF), freedom from metastases, and survival. RESULTS: Men in arm 1 exhibited a greater incidence of low-grade influenza-like symptoms, transaminitis, neutropenia, and thrombocytopenia than men in arm 2. There were no significant differences in gastrointestinal or genitourinary events or QOL between the 2 arms. Two-year prostate biopsies were obtained from 37 men (84%). Thirty-three percent of men in arm 1 were biopsy-positive versus 58% in arm 2, representing a 42% relative reduction in biopsy positivity in the investigational arm (P=.13). There was a 60% relative reduction in biopsy positivity in the investigational arm in men with <50% positive biopsy cores at baseline (P=.07). To date, 1 patient in each arm exhibited biochemical failure (arm 1, 4.8%; arm 2, 4.3%). No patient developed hormone-refractory or metastatic disease, and none has died from prostate cancer. CONCLUSIONS: Combining OAMCGT with IMRT does not exacerbate the most common side effects of prostate radiation therapy and suggests a clinically meaningful reduction in positive biopsy results at 2 years in men with intermediate-risk prostate cancer.

Feasibility of adenovirus-mediated hNIS gene transfer and 131I radioiodine therapy as a definitive treatment for localized prostate cancer.

We have developed a replication-competent adenovirus (Ad5-yCD/mutTK(SR39)rep-hNIS) armed with two suicide genes and the human sodium iodide symporter (hNIS) gene. In this context, hNIS can be used as a reporter gene in conjunction with nuclear imaging and as a potentially therapeutic gene when combined with (131)I radioiodine therapy. Here, we quantified the volume and magnitude of hNIS gene expression in the human prostate following injection of a high Ad5-yCD/mutTK(SR39)rep-hNIS dose using a standardized injection algorithm, and estimated the radiation dose that would be delivered to the prostate had men been administered (131)I with curative intent. Six men with clinically localized prostate cancer received an intraprostatic injection of Ad5-yCD/mutTK(SR39)rep-hNIS under transrectal ultrasound guidance. All men received 2 × 0.5 ml deposits (5 × 10(11) vp/deposit) in each of the four base and midgland sextants and 2 × 0.25 ml deposits (2.5 × 10(11) vp/deposit) in each of the two apex sextants for a total of 12 deposits (5 × 10(12) vp) in 5 ml. On multiple days after the adenovirus injection, men were administered sodium pertechnetate (Na(99m)TcO(4)) and hNIS gene expression in the prostate was quantified by single photon emission computed tomography (SPECT). hNIS gene expression was detected in the prostate of six of six (100%) men. On average, 45% (range 18-83%) of the prostate volume was covered with gene expression. Had men been administered 200 mCi (131)I, we estimate that the mean absorbed dose to the prostate would be 7.2 ± 4.8 Gy (range 2.1-13.3 Gy), well below that needed to sterilize the prostate. We discuss the obstacles that must be overcome before adenovirus-mediated hNIS gene transfer and (131)I radioiodine therapy can be used as a definitive treatment for localized prostate cancer.

Adaptive seamless design for an efficacy trial of replication-competent adenovirus-mediated suicide gene therapy and radiation in newly-diagnosed prostate cancer (ReCAP Trial).

PURPOSE: Cumulative evidence has suggested investigation of the efficacy of Replication-Competent Adenovirus-mediated Suicide Gene Therapy in newly-diagnosed Prostate Cancer (ReCAP). There is a challenge in designing an efficacy trial for newly-diagnosed prostate cancer given its long natural history. The regulatory agency recommended a Phase II trial for safety before conducting the efficacy trial. EXPERIMENTAL DESIGN: The ReCAP trial is an adaptive seamless, multi-site open-label, randomized Phase II/III trial. Two hundred eighty men will be randomized to receive either replication-competent adenovirus-mediated suicide gene therapy followed by radiation (Arm 1) or radiation alone (Arm 2). Phase II trial component will include the first 21 patients in Arm 1 with complete toxicity through day 90 for safety evaluation. The primary efficacy endpoint is the time free from biochemical and/or clinical failure (FFF). The secondary efficacy endpoints are 2-year prostate biopsies and overall survival. Unequal spaced interim looks are proposed with the adaptive sample-size re-estimation. RESULTS: This trial has been approved by the FDA for the study therapy investigation and is currently recruiting patients. CONCLUSIONS: Challenges remain in designing newly-diagnosed prostate cancer trials. Adaptive seamless design is time-saving and a cost-effective design in the development of novel medical therapies, but requires a specified statistical plan in the decision process involved.

A novel method of boron delivery using sodium iodide symporter for boron neutron capture therapy.

Boron Neutron Capture Therapy (BNCT) effectiveness depends on the preferential sequestration of boron in cancer cells relative to normal tissue cells. We present a novel strategy for sequestering boron using an adenovirus expressing the sodium iodide symporter (NIS). Human glioma grown subcutaneously in athymic mice and orthotopic rat brain tumors were transfected with NIS using a direct tumor injection of adenovirus. Boron bound as sodium tetrafluoroborate (NaBF(4)) was administered systemically several days after transfection. Tumors were excised hours later and assessed for boron concentration using inductively coupled plasma atomic emission spectroscopy. In the human glioma transfected with NIS, boron concentration was more than 10 fold higher with 100 mg/kg of NaBF(4), compared to tumor not transfected. In the orthotopic tumor model, the presence of NIS conferred almost 4 times the boron concentration in rat tumors transfected with human virus compared with contralateral normal brain not transfected. We conclude that adenovirus expressing NIS has the potential to be used as a novel boron delivery agent and should be explored for future clinical applications.

Does hyperthermia increase adenoviral transgene expression or dissemination in tumors?

PURPOSE: Viral vectors used for cancer gene therapy are usually delivered by direct intratumoral administration. We studied the role of hyperthermia (HT) in vitro and in vivo in an attempt to achieve higher transfection rates (especially, larger volume of spread). MATERIALS AND METHODS: Replication-deficient adenoviruses containing either the human sodium-iodide symporter (Ad5-CMV-hNIS) or green fluorescent protein (Ad5-CMV-eGFP) as reporter genes were used. For in vitro studies, human lung cancer A549 cells were transfected with the virus and assayed for hNIS expression by radioactive pertechnetate uptake or green fluorescence activity using a gamma-counter or fluoroscopy respectively in the presence and absence of HT. For in vivo studies, A549 tumors were established intramuscularly in CD1 athymic mice. The adenoviral constructs (10(10) viral particles/tumor) were injected intratumorally when the tumors reached 10-11 mm in diameter. Different timing sequences of HT were examined and viral spread was assessed using technetium-autoradiography or GFP-fluorescence microscopy. RESULTS: In the in vitro studies, A549 cells infected with the adenoviral construct did not show any difference in gene expression level in the presence or absence of HT. In vivo, the effect of HT on the volume of gene expression in A549 tumors was highly variable with some groups of mice showing better spread in the presence of HT and others showing reduced spread with HT. CONCLUSION: Improvements in intratumoral adenoviral spread in response to hyperthermia were not consistently observed in a mouse tumor model using two quantitative endpoints of gene expression.

Phase I study of noninvasive imaging of adenovirus-mediated gene expression in the human prostate.

To monitor noninvasively potentially therapeutic adenoviruses for cancer, we have developed a methodology based on the sodium iodide symporter (NIS). Men with clinically localized prostate cancer were administered an intraprostatic injection of a replication-competent adenovirus, Ad5-yCD/utTK(SR39)rep-hNIS, armed with two suicide genes and the NIS gene. NIS gene expression (GE) was imaged noninvasively by uptake of Na(99 m)TcO(4) in infected cells using single photon emission-computed tomography (SPECT). The investigational therapy was safe with 98% of the adverse events being grade 1 or 2. GE was detected in the prostate in seven of nine (78%) patients at 1 x 10(12) virus particles (vp) but not at 1 x 10(11) vp. Volume and total amount of GE was quantified by SPECT. Following injection of 1 x 10(12) vp in 1 cm(3), GE volume (GEV) increased to a mean of 6.6 cm(3), representing, on average, 18% of the total prostate volume. GEV and intensity peaked 1-2 days after the adenovirus injection and was detectable in the prostate up to 7 days. Whole-body imaging demonstrated intraprostatic gene expression, and there was no evidence of extraprostatic dissemination of the adenovirus by SPECT imaging. The results demonstrate that noninvasive imaging of adenovirus-mediated gene therapy in humans is feasible and safe.

Mitigation of radiation-induced skin injury by AAV2-mediated MnSOD gene therapy.

BACKGROUND: Radiation-induced, long-lived free radicals, reactive oxygen species and pro-inflammatory cytokines have been implicated in the resultant tissue injury after exposure to ionizing radiation. METHODS: An approach designed to reduce the damaging effects of reactive oxidants employs metalloenzymes of superoxide dismutase (SOD), such as MnSOD. Recombinant adeno-associated virus 2 (AAV2) provides safe and long-term expression in humans. We tested the effectiveness of AAV2-MnSOD-hrGFP, a vector expressing MnSOD and green fluorescent protein (GFP) in preclinical models. RESULTS: Infection of cultured cells with AAV2-MnSOD-hrGFP showed enhanced expression of MnSOD and GFP. Sustained expression of GFP was achieved for at least 1 month in vivo following administration of AAV2-MnSOD-hrGFP to subcutaneous tissue of C57BL/6J mice. A single subcutaneous injection of AAV2-MnSOD-hrGFP significantly mitigated acute skin injury following single dose of irradiation of either 30 or 35 Gy. CONCLUSIONS: The proof-of-concept demonstrated in the present study together with the known safety profile in humans indicate that AAV-mediated MnSOD expression has potential countermeasure utility against normal tissue injury following radiation therapy or radiological accident.

Reporter gene imaging using radiographic contrast from nonradioactive iodide sequestered by the sodium-iodide symporter.

The hypothesis that the human sodium-iodide symporter, NIS, can be used to detect NIS expression using standard radiological techniques was tested using adenoviral transduced NIS expression in human tumor xenografts grown in mice and in a naive dog prostate. Nonradioactive iodide was administered systemically to animals that 1-3 days previously had received a local injection of a replication-competent adenovirus expressing NIS under the control of the CMV promoter. The distribution of radiopacity was assessed in mouse tumors using micro-CT and a clinical X-ray machine and in the prostate of an anesthetized dog using a clinical spiral CT. Iodide sequestration and NIS expression were measured using X-ray spectrochemical analysis and fluorescence microscopy, respectively. Radiographic contrast due to NIS gene expression that was observed indicates the technique has potential for use in preclinical rodent tumor studies but probably lacks sensitivity for human use.

A model for optimizing adenoviral delivery in human cancer gene therapy trials.

Optimization of adenoviral delivery to the target volume is required for adenovirus-mediated cancer gene therapy to reach its maximal potential. The purpose of these studies was to develop a model of gene expression to improve adenovirus-mediated cancer gene therapy in the clinic. We measured the distribution of gene expression after a single deposit of a replication-competent adenovirus carrying the human sodium iodide symporter (hNIS) reporter gene was delivered to naive canine prostate and to human tumor xenografts. We generated hypothetical treatment plans for two prospective prostate cancer patients, using standard brachytherapy algorithms. In both models, the gene expression distribution from a single adenoviral deposit could be accurately described by a Gaussian function. In the naive canine prostate, a 0.1-ml deposit of 3 x 10(11) viral particles (VP) resulted in a gene expression volume of 1.14 +/- 0.70 cm(3), indicating that a minimum of 40 adenoviral deposits would be required to cover a 40-cm(3) prostate with therapeutic gene expression. On a viral particle basis, the gene expression volume obtained in human tumor xenografts (7 x 10(-12) cm(3)/VP) was twice that (3.5 x 10(-12) cm(3)/VP) measured in the naive canine prostate. Hypothetical treatment plans for two prostates indicated that 26 and 57 0.1-ml adenoviral deposits would be required to cover, respectively, 24- and 49-cm(3) prostates with gene expression. Although our studies focused on prostate, we believe the methodology to model gene expression presented here has much broader application to optimize treatment plans in other solid tumor sites; this assertion should be confirmed experimentally.

Replication-competent adenovirus-mediated suicide gene therapy with radiation in a preclinical model of pancreatic cancer.

In preparation for a Phase I trial, we evaluated the efficacy and toxicity of replication-competent adenovirus-mediated suicide gene therapy in combination with radiation in a preclinical model of pancreatic cancer. Human MiaPaCa-2 and PANC-1 pancreatic adenocarcinoma cells were found to be sensitive to the oncolytic effects of the Ad5-yCD/mutTK(SR39)rep-ADP adenovirus and also to the cytotoxic effects of the yeast cytosine deaminase (yCD) and herpes simplex virus thymidine kinase (HSV-1 TK(SR39)) genes in vitro. Combining Ad5-yCD/mutTK(SR39)rep-ADP-mediated suicide gene therapy with radiation significantly increased tumor control beyond that of either modality alone. Injection of Ad5-yCD/mutTK(SR39)rep-ADP in the dog pancreas at doses (10(12) virus particle (vp)) to be used in humans resulted in mild pancreatitis but not peritonitis or hepatotoxicity. Following administration of 9-(4-[(18)F]-fluoro-3-hydroxymethylbutyl)guanine ([(18)F]-FHBG), a positron-emitting substrate of HSV-1 TK, Ad5-yCD/mutTK(SR39)rep-ADP activity could be monitored non-invasively by positron emission tomography (PET). [(18)F]-FHBG uptake was readily detected in the pancreas but not in other major abdominal organs, indicating that little of the injected adenovirus disseminates to collateral tissues. These results demonstrate that Ad5-yCD/mutTK(SR39)rep-ADP-mediated suicide gene therapy has the potential to augment the effectiveness of pancreatic radiotherapy without resulting in excessive toxicity. Hence they provide the scientific basis for an ongoing Phase I trial in pancreatic cancer.

Design considerations for incorporating sodium iodide symporter reporter gene imaging into prostate cancer gene therapy trials.

This study was done to aid in the design of a phase I gene therapy trial in patients with prostate cancer. We determined the dosimetric characteristics of our reporter gene system when coupled with intravenous administration of radioactive sodium pertechnetate (Na(99m) TcO(4)) and determined the feasibility of using human sodium iodide symporter (hNIS) as a reporter gene to study the dynamics of adenoviral transgene expression in a large animal tumor. A replication-competent Ad5-yCD/mutTK(SR39) rep-hNIS adenovirus was injected into the prostate gland of dogs for dosimetry purposes, and into a canine soft tissue sarcoma (STS) for imaging purposes. After resection of the prostate, the amount of (99m)TcO(4)() sequestered in the prostate was determined, the radiation dose absorbed by the prostate and nontarget critical organs was calculated, and hNIS reporter gene expression was imaged in the STS by single-photon emission computed tomography (SPECT). On the basis of the findings from 25 dogs, the amount of (99m)TcO (4)() sequestered in the prostate ranged from 13 to 276 muCi. Using the highest value observed, absorbed radiation dose to critical organs was calculated and found to be below U.S. Food and Drug Administration limits for diagnostic imaging. Also, (99m)TcO (4)() uptake was readily detected by SPECT and found to persist in vivo for at least 4 days. On the basis of our dosimetry calculations, up to five imaging procedures can be safely performed in humans after intraprostatic injection of the Ad5-yCD/mutTK(SR39)rep-hNIS adenovirus and the hNIS reporter gene system can be used to study the dynamics of adenoviral gene therapy vectors in large animal tumors.

Prostate cancer gene therapy clinical trials.

Despite recent advances in early detection and treatment, prostate cancer is still the second leading cause of cancer death in men in the United States, and approximately 27,000 men will die from it this year. Better treatments are needed for aggressive forms of localized disease and hormone-refractory metastatic disease. Recently, several gene therapy strategies have generated provocative results in early-stage clinical trials, raising the possibility that gene therapy may have the potential to affect both localized and metastatic disease. Much work lies ahead. Nevertheless, for the time being, these studies provide hope that gene therapy may someday earn a place in the management of prostate cancer.

Phase I trial of replication-competent adenovirus-mediated suicide gene therapy combined with IMRT for prostate cancer.

Replication-competent adenovirus-mediated suicide gene therapy is an investigational cancer treatment in which an oncolytic adenovirus armed with chemo-radiosensitizing genes is used to destroy tumor cells. Previously, we evaluated the toxicity and efficacy of this approach in two clinical trials of prostate cancer using a first-generation adenovirus. Here, we report the toxicity and preliminary efficacy of this approach in combination with intensity-modulated radiotherapy (IMRT) in patients with newly diagnosed prostate cancer using an improved, second-generation adenovirus. The investigational therapy was associated with low toxicity, and there were no dose-limiting toxicities or treatment-related serious adverse events. Relative to a previous trial using a first-generation adenovirus, there was no increase in hematologic, hepatic, gastrointestinal (GI), or genitourinary (GU) toxicities. Post-treatment prostate biopsies yielded provocative preliminary results. When the results of two similar trials were combined, 22% of evaluable patients were positive for adenocarcinoma at their last biopsy, which is better than expected (>or=40%) for this cohort of patients (P=0.038). When the results were categorized by prognostic risk, most of the treatment effect was observed in the intermediate-risk group, with 0 of 12 patients (0%) being positive for cancer at their last biopsy (P<0.01). These results further demonstrate the safety of this investigational approach and raise the possibility that it may have the potential to improve the outcome of conformal radiotherapy in select patient groups.

Five-year follow-up of trial of replication-competent adenovirus-mediated suicide gene therapy for treatment of prostate cancer.

Replication-competent adenovirus-mediated suicide gene therapy is an investigational cancer treatment that combines the oncolytic actions of human adenoviruses with the cytotoxic effects of chemo-radiosensitizing genes. Previously, we reported the short-term effects of this therapy in men with local recurrence of prostate cancer after definitive radiotherapy. With a median prostate-specific antigen (PSA) follow-up of 5 years, we report here the effect of the gene therapy on prostate-specific antigen doubling time (PSADT), a surrogate end point with significant prognostic power. When considering all evaluable subjects, the PSADT increased following the gene therapy from a mean of 17 to 31 months (median 16 to 22 months) (P=0.014). Assuming that salvage androgen suppression therapy androgen suppression therapy (AST) was uniformly initiated at a PSA of 15 ng/mL, the gene therapy would have delayed the projected onset of salvage therapy by an average of 2 years. The results indicate that replication-competent adenovirus-mediated suicide gene therapy may provide a potential long-term benefit to patients, as shown by a lengthening of the PSADT, and delay in when salvage therapy is indicated. Given the high morbidity associated with AST, we believe this approach could provide an attractive treatment option for selection of patients experiencing PSA relapse following definitive therapy.

A novel mechanism of synergistic cytotoxicity with 5-fluorocytosine and ganciclovir in double suicide gene therapy.

The combination of cytosine deaminase (CD) and herpes simplex virus thymidine kinase (HSV-TK) suicide gene protocols has resulted in enhanced antitumor activity in cultured tumor cells and animal models. In this study, we show that concurrent addition of prodrugs 5-fluorocytosine (5-FC) and ganciclovir (GCV) was less efficacious than sequential treatment in human DU145 prostate carcinoma cells infected with an adenovirus containing a CD/HSV-TK fusion gene. If cells were incubated for 24 hours with 5-FC followed by a 24-hour GCV treatment, GCV triphosphate levels were 2-fold higher, incorporation of GCV monophosphate into DNA was 2.5-fold higher, and growth inhibition was increased 4-fold compared with simultaneous treatment. As expected, cellular dTTP levels were reduced during the 5-FC preincubation. However, dGTP pools also declined parallel to the dTTP decrease. Similar results were obtained when 5-fluorouracil or 5-fluoro-2'-deoxyuridine was used instead of CD/5-FC. These data allowed us to propose a novel hypothesis for the synergistic interaction between CD/5-FC and HSV-TK/GCV treatments. We suggest that the CD/5-FC-mediated reduction of dTTP results in a concurrent decrease of dGTP due to allosteric regulation of ribonucleotide reductase. Because dGTP is the endogenous competitor of GCV triphosphate, depleted dGTP at the time of GCV addition results in increased GCV in DNA and cell kill. In fact, addition of deoxyguanosine during the 5-FC incubation reverses the dGTP depletion, reduces the amount of GCV monophosphate incorporated into DNA, and prevents the CD/5-FC-mediated enhancement of HSV-TK/GCV cytotoxicity. Understanding this mechanistic interaction may help recognize better strategies for creating more efficacious clinical protocols.

Second-generation replication-competent oncolytic adenovirus armed with improved suicide genes and ADP gene demonstrates greater efficacy without increased toxicity.

Replication-competent adenovirus-mediated suicide gene therapy has proven to be safe in humans when delivered intraprostatically. Although signs of efficacy are emerging, it is likely that further improvements will be needed before this technology will have widespread applicability in the clinic. Toward this end, we have developed a second-generation, replication-competent adenovirus (Ad5-yCD/mutTK(SR39)rep-ADP) containing an improved yeast cytosine deaminase (yCD)/mutant(SR39) herpes simplex virus thymidine kinase fusion (yCD/mutTK(SR39)) gene and the adenovirus death protein (ADP) gene. Relative to the first-generation Ad5-CD/TKrep adenovirus, Ad5-yCD/mutTK(SR39)rep-ADP demonstrated greater tumor cell kill in vitro and significantly greater tumor control in preclinical models of human cancer. Quantification of transgene volume following direct injection of fadenovirus into human tumor xenografts and the naïve canine prostate demonstrated that ADP enhanced adenoviral spread in vivo. Toxicology studies were performed to determine whether the improved yCD/mutTK(SR39) fusion and ADP genes increased toxicity. Intraprostatic injection of Ad5-yCD/mutTK(SR39)rep-ADP did not result in significantly increased toxicity relative to the parental Ad5-CD/TKrep adenovirus, the latter of which has proven to be safe in two Phase I prostate cancer clinical trials. Together, these results provide the scientific basis for evaluating the safety and efficacy of the second-generation Ad5-yCD/mutTK(SR39)rep-ADP adenovirus in humans.