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At least 451 records · Page 25Linked to original sources

PubMed1995

Cancer gene therapy using plasmid DNA: purification of DNA for human clinical trials.

A production method has been developed for the purification of pharmaceutical-grade plasmid DNA for in vivo gene therapy. This method has been applied to the purification of VCL-1005, which is a eukaryotic plasmid expression vector that codes for the production of the HLA-B7 protein. Purified VCL-1005 is formulated with a cationic lipid and injected directly into established tumors of HLA-B7-negative patients with advanced cancers to heighten the patient's immune response against the cancer. The purification of pharmaceutical-grade plasmid DNA requires the development of highly reproducible and scaleable processing methods that meet regulatory standards similar to those required for the manufacture of recombinant protein pharmaceuticals. Defined pharmaceutical standards of purity, potency, efficacy, and safety are routinely met by the process described in this study. The scaleable purification method described here is a combination of highly reproducible unit operations; alkaline lysis, precipitation, and size-exclusion chromatography. The advantages over existing DNA purification methods include improved plasmid purity and the elimination of undesirable process additives such as toxic organic extractants and animal-derived enzymes. The overall process yield of purified plasmid DNA from fermentation through final column purified product is greater than 50%. Contaminating Escherichia coli DNA levels are reproducibly below 1% as measured by Southern analysis. Endotoxin levels are less than 0.03 endotoxin units/micrograms plasmid DNA and residual protein is undetectable. This process was used to produce 100 mg of VCL-1005 for use in an active clinical protocol.

Blotting, Southern↗

PubMed2002

Hypermutable bases in the p53 cancer gene are at vulnerable positions in DNA secondary structures.

A DNA folding analysis indicates that the most hypermutable bases in exons 5, 7, and 8 of the p53 tumor suppressor gene are located immediately next to stems in stable DNA stem-loop structures. On the basis of the highest negative energy (-DeltaG) value of the structures containing each mutable bases and on the extent to which each base is unpaired during transcription, their relative mutabilities are calculated using a new computer algorithm. These predicted mutation frequencies correlate well with those observed in 14,000 human cancers (R(2) = 0.76), whereas there is no such correlation (R(2) = 0.0005) for nearby control bases. The correlation of hypermutable base frequencies with -DeltaG values is poor (R(2) = 0.19), indicating that the extent to which a base is unpaired during transcription is a significant contribution to predicting mutation frequencies.

Bacterial Proteins↗

PubMed2006

Immuno-isolation in cancer gene therapy.

The implantation of genetically-modified non-autologous cells in immuno-protected microcapsules is an alternative to ex vivo gene therapy. Such cells delivering a recombinant therapeutic product are isolated from the host's immune system by being encapsulated within permselective microcapsules. This approach has been successful in pre-clinical animal studies involving delivery of hormone or enzymes to treat dwarfism, lysosomal storage disease, or hemophilia B. Recently, this platform technology has shown promise in the treatment for more complex diseases such as cancer. One of the earliest strategy was to augment the chemotherapeutic effect of a prodrug by implanting encapsulated cells that can metabolise prodrugs into cytotoxic products in close proximity to the cancer cells. More recent approaches include enhancing tumor cell death through immunotherapy, or suppressing tumor cell proliferation through anti-angiogenesis. These can be achieved by delivering single molecules of cytokines or angiostatin, respectively, by implanting microencapsulated cells engineered to secrete these recombinant products. Recent refinements of these approaches include genetic fusion of cytokines or angiostatin to additional functional groups with tumor targeting or tumor cell killing properties, thus enhancing the potency of the recombinant products. Furthermore, a COMBO strategy of implanting microencapsulated cells to deliver multiple products targeted to diverse pathways in tumor suppression also showed much promise. This review will summarise the application of microencapsulation of genetically-modified cells to cancer treatment in animal models, the efficacy of such approaches, and how these studies have led to better understanding of the biology of cancer treatment. The flexibility of this modular system involving molecular engineering, cellular genetic modification, and polymer chemistry provides potentially a huge range of application modalities, and a tremendous multi-disciplinary challenge for the future.

Animals↗

PubMed2006

Suicide cancer gene therapy using pore-forming toxin, streptolysin O.

We cloned the streptolysin O gene from the Streptococcus pyogenes genome and tested the possibility of using it as an anticancer reagent. Transient transfection of the streptolysin O gene efficiently killed 293T cells after 12 hours of transfection as determined by lactate dehydrogenase release and propidium iodide uptake. No caspase activity was observed and necrosis was prominent during streptolysin O-induced cell death. Biochemical analysis of streptolysin O protein revealed that the deletion of only 5 amino acids from the COOH-terminal region of streptolysin O, which is essential for cholesterol binding activity, abolished its cell-killing activity, whereas the NH2-terminal region was more resilient, i.e., up to 115 amino acids could be deleted without changing its cell-killing activity. We generated a streptolysin O-expressing adenovirus and injected it into human cervical cancer cell-derived tumors grown in a nude mouse model. Twenty-one days postinjection, the average size of tumors in the streptolysin O adenovirus-injected group was 29.3% of that of the control PBS-treated group. Our results show that the genes of pore-forming toxins, like streptolysin O protein, have the potential to establish a novel class of suicide gene therapeutic reagents.

Adenoviridae↗

PubMed2005

Antiangiogenic cancer gene therapy by adeno-associated virus 2-mediated stable expression of the soluble FMS-like tyrosine kinase-1 receptor.

Antiangiogenic gene transfer has the potential to be more efficacious than protein-based therapies or pharmacotherapies for the control of solid tumor growth, invasion and metastasis. For a sustained antiangiogenic effect, a vector capable of long-term expression without vector-associated immunity or toxicity is advantageous. The present study evaluated the potential of a recombinant adeno-associated virus-2 (rAAV) encoding the human soluble FMS-like tyrosine kinase receptor 1 (sFlt-1), which functions by both sequestering vascular endothelial growth factor (VEGF) and forming inactive heterodimers with other membrane-spanning VEGF receptors, in vitro and in vivo. Results indicated significant growth inhibitory activity of the transgenic factor in a human umbilical vein endothelial cell proliferation assay in vitro and protection against the growth of an angiogenesis-dependent human ovarian cancer cell line, SKOV3.ip1, xenograft in vivo with increased disease-free survival. Stable expression of the secretory factor and transgene persistence were confirmed by immunohistochemistry and in situ hybridization analyses, respectively. Increased therapeutic effects on both the growth index of the implanted tumor cells and tumor-free survival also correlated with an increasing dose of the vector used. These studies indicate that rAAV-mediated sFlt-1 gene therapy may be a feasible approach for inhibiting tumor angiogenesis, particularly as an adjuvant/therapy.

Angiogenesis Inhibitors↗

PubMed2000

Tumor-specific transgene expression from the human telomerase reverse transcriptase promoter enables targeting of the therapeutic effects of the Bax gene to cancers.

Human telomerase reverse transcriptase (hTERT) is the catalytic subunit of telomerase, which is highly active in immortalized cells and >85% of human cancers but is quiescent in most normal somatic cells. To test the feasibility of using the hTERT promoter to induce tumor-specific transgene expression in cancer gene therapy, we constructed an adenoviral vector expressing a LacZ reporter gene driven by the hTERT core promoter and evaluated its activity in vitro and in vivo. The hTERT promoter could drive high-level expression of LacZ in tumor cells but not in normal cells and normal mouse tissues. Using a binary adenoviral system that can induce Bax gene expression, we showed that induction of the Bax gene expression via the hTERT promoter elicited tumor-specific apoptosis in vitro, suppressed tumor growth in nude mice, and prevented the toxicity of the Bax gene in vitro and in vivo. Thus, the hTERT promoter is apparently a strong and tumor-selective promoter with potential application in targeted cancer gene therapy.

Adenoviridae↗

PubMed2001

Studies on human colon cancer gene APC by targeted expression in Drosophila.

Mutations in human Adenomatous Polyposis Coli (APC) gene are associated with both familial and sporadic colorectal tumors. APC is known to down regulate beta-catenin levels, a transducer of Wnt signaling. The aim of this study is to provide transgenic Drosophila expressing either full-length or truncated forms of human APC (hAPC) protein and methods for using them in functional genomics and drug screening. Consistent with its biochemical properties, targeted expression of either full-length hAPC or its beta-catenin binding domain alone negatively regulated the function of the beta-catenin homologue, Armadillo (Arm) and thereby, inhibited Wnt/Wg signaling during fly development. hAPC inhibited Arm function even in the absence of GSK-3beta activity, although the latter was required to mediate the degradation of Arm. Consistent with this, hAPC suppressed the phenotypes induced by the over-expression of degradation-resistant forms of Arm. Subsequently, using hAPC-induced eye phenotypes as the assay in a suppressor-enhancer screen, we have identified two new loci in Drosophila, which modulate Wnt/Wg signaling. In addition, an anti-colon cancer drug, indomethacin, specifically enhanced hAPC-induced phenotypes.

Adenomatous Polyposis Coli Protein↗

PubMed2004

Cancer gene therapy through autonomous parvovirus--mediated gene transfer.

Parvoviruses are small nuclear replicating DNA viruses. The rodent parvoviruses are usually weakly pathogenic in adult animals, bind to cell surface receptors which are fairly ubiquitously expressed on cells, and do not appear to integrate into host chromosomes during either lytic or persistent infection. The closely related rodent parvoviruses MVM, H-1 and LuIII efficiently infect human cell lines. Most interesting, malignant transformation of human and rodent cells was often found to correlate with a greater susceptibility to parvovirus-induced killing (oncolysis) and with an increase in the cellular capacity for amplifying and / or expressing the incoming parvoviral DNA. These and other interesting properties make these autonomous rodent parvoviruses and recombinant derivatives promising candidate antitumor vectors. Capsid replacement vectors have been produced from MVM or H-1 virus that carry transgenes encoding either therapeutic products (cytokines/chemokines, Apoptin, herpes simplex virus thymidine kinase) or marker proteins (green fluorescent protein, chloramphenicolacetyl transferase, luciferase). This review describes the current state of the art regarding the potential application of wild-type parvoviruses and derived vectors for the treatment of cancer. In particular, recent successes with the development of replication-competent virus-free vector stocks are discussed and results from pre-clinical studies using recombinant parvoviruses transducing various cytokines/chemokines are presented.

Genetic Therapy↗

PubMed2001

Chimeric PSA enhancers exhibit augmented activity in prostate cancer gene therapy vectors.

The native PSA enhancer and promoter confer prostate-specific expression when inserted into adenovirus vectors capable of efficient in vivo gene delivery, although the transcriptional activity is low. By exploiting properties of the natural PSA control regions, we have improved the activity and specificity of the prostate-specific PSA enhancer for gene therapy and imaging applications. Previous studies have established that androgen receptor (AR) molecules bind cooperatively to AREs in the PSA enhancer core (-4326 to -3935) and act synergistically with AR bound to the proximal promoter to regulate transcriptional output. To exploit the synergistic nature of AR action we generated chimeric enhancer constructs by (1) insertion of four tandem copies of the proximal AREI element; (2) duplication of enhancer core; or (3) removal of intervening sequences (-3744 to -2855) between the enhancer and promoter. By comparing to the baseline construct, PSE, containing the PSA enhancer (-5322 to -2855) fused to the proximal promoter (-541 to +12), the three most efficacious chimeric constructs, PSE-BA (insertion of ARE4), PSE-BC (duplication of core) and PSE-BAC (insertion of core and ARE4), are 7.3-, 18.9-, and 9.4-fold higher, respectively. These chimeric PSA enhancer constructs are highly androgen inducible and retain a high degree of tissue discriminatory capability. Initial biochemical studies reveal that the augmented activity of the chimeric constructs in vivo correlates with their ability to recruit AR and critical co-activators in vitro. The enhanced activity, inducibility and specificity of the chimeric constructs are retained in an adenoviral vector (Ad-PSE-BC-luc). Systemic administration of Ad-PSE-BC-luc into SCID mice harboring the LAPC-9 human prostate cancer xenografts shows that this prostate specific vector retained tissue discriminatory capability compared with a comparable cytomegalovirus (CMV) promoter driven vector.

Adenoviridae↗

PubMed2002

Apoptotic genes in cancer therapy.

Induction of apoptosis in malignant cells is a major goal of cancer therapy in general and of certain cancer gene therapy strategies in particular. Numerous apoptosis-regulating genes have been evaluated for this purpose. Besides the most prominent p53 gene others include p16, p21, p27, E2F genes, FHIT, PTEN and CASPASE genes. Recently, the potential for therapy of an adenoviral gene, E1A, known for a long time for its apoptosis-inducing activity, has been discovered. In experimental settings, these genes have proven their tumor-suppressive and apoptosis-inducing activity. Clinical trials are currently being performed with selected genes. By far the most studies transfer the p53 gene using retro- or adenoviral vectors. Disease stabilization or other benefits were observed in a limited number of patients when p53 was applied alone or in combination with cytotoxic drugs. A second proapoptotic gene that has entered clinical trials is adenovirus E1A. Here, too, disease stabilization as well as/or local regression in one case have been demonstrated in selected patients. In all cases, side effects were tolerable. To further improve E1A as a therapeutic transgene, we have deleted transforming domains from the adenovirus 5 and 12 13S cDNAs. Mutants were derived which had completely lost their transforming activity in combination with the E1B oncogene but retained a pronounced tumor-suppressive activity. Cells transduced with these constructs showed a highly reduced ability to grow in soft agar, and tumor growth in nude mice could be substantially suppressed. Outgrowing tumors had lost E1A expression when analyzed in Western blots. These E1A constructs may represent valuable tools for cancer gene therapy in the future.

Adenoviridae↗

PubMed2000

Testing adolescents for a hereditary breast cancer gene (BRCA1): respecting their autonomy is in their best interest.

The testing of individuals before the age of 18 years for hereditary late-onset diseases has been judged ethically not acceptable in guidelines and directives published by medical professional organizations. However, there are not enough best interest arguments to deny genetic testing to an adolescent at risk of carrying a BRCA1 mutation, even if the competence of adolescents for medical decisions is considered to be lower than the competence of adults. The adolescent's decision is not irrational or of very high risk. Respecting adolescents' autonomous choices concerning genetic testing has positive consequences for their self-esteem and psychological health. Geneticists and other professionals should clearly differentiate between children and adolescents in regard to BRCA1 gene testing and recommend giving decision autonomy about the test to all psychologically "normal" adolescents.

Adolescent↗

PubMed2004