ABSTRACT
Vectors mediating strong, durable, and tissue-specific transgene expression are mandatory for safe and effective gene therapy. In settings requiring systemic vector administration, the availability of suited vectors is extremely limited. Here, we present a strategy to select vectors with true specificity for a target tissue from random peptide libraries displayed on adeno-associated virus (AAV) by screening the library under circulation conditions in a murine model. Guiding the in vivo screening by next-generation sequencing, we were able to monitor the selection kinetics and to determine the right time point to discontinue the screening process. The establishment of different rating scores enabled us to identify the most specifically enriched AAV capsid candidates. As proof of concept, a capsid variant was selected that specifically and very efficiently delivers genes to the endothelium of the pulmonary vasculature after intravenous administration. This technical approach of selecting target-specific vectors in vivo is applicable to any given tissue of interest and therefore has broad implications in translational research and medicine.
Subject(s)
Capsid/metabolism , Dependovirus/genetics , High-Throughput Nucleotide Sequencing/methods , Lung/metabolism , Animals , Capsid Proteins/genetics , Capsid Proteins/metabolism , Dependovirus/metabolism , Genetic Therapy , Genetic Vectors/administration & dosage , Mice , Organ Specificity , Peptide Library , Transduction, GeneticABSTRACT
Prostate cancer (PCa) is the most commonly diagnosed type of cancer in men in western industrialized countries. As a public health burden, the need for the invention of new cost-saving PCa immunotherapies is apparent. In this study, we present a DNA vaccine encoding for the prostate-specific antigen prostatic acid phosphatase (PAP) linked to the J-domain and the SV40 enhancer sequence. The PAP DNA vaccine induced a strong PAP-specific cellular immune response after electroporation (EP)-based delivery in C57BL/6 mice. Splenocytes from mice immunized with PAP recognized the naturally processed PAP epitopes, indicating that vaccination with the PAP-J gene broke its self-tolerance against PAP. Remarkably, DNA vaccination with PAP-J inhibited tumor growth in the Transgenic Adenocarcinoma of the Mouse Prostate (TRAMP) mouse model that closely resembled human PCa. Therefore, this study highlights a novel cancer immunotherapy approach with the potential to control PCa in clinical settings.
Subject(s)
Prostatic Neoplasms/therapy , Protein Tyrosine Phosphatases/genetics , Self Tolerance/genetics , Acid Phosphatase , Amino Acid Motifs , Amino Acid Sequence , Animals , Antibodies/immunology , Antigens, Neoplasm/chemistry , Antigens, Neoplasm/genetics , Antigens, Neoplasm/immunology , Cancer Vaccines/genetics , Cancer Vaccines/immunology , Cell Line , Disease Models, Animal , Disease Progression , Epitopes/immunology , Epitopes/metabolism , Genetic Vectors/genetics , H-2 Antigens/immunology , H-2 Antigens/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Peptides/immunology , Peptides/metabolism , Prostatic Neoplasms/genetics , Prostatic Neoplasms/immunology , Protein Binding , Protein Tyrosine Phosphatases/chemistry , Protein Tyrosine Phosphatases/immunology , T-Lymphocytes, Cytotoxic/immunology , Vaccines, DNA/immunologyABSTRACT
Therapeutic DNA vaccination is an attractive adjuvant option to conventional methods in the fight against cancer, like surgery radiotherapy and chemotherapy. Despite strong antitumor effects that were observed in small animals with different antigens, DNA-based vaccines remain weakly immunogenic in large animals and primates compared to protein-based vaccines. Here, we sought to enhance the immunogenicity of a therapeutic nontransforming cervical cancer DNA vaccine (HPV-16 E7SH) by introduction of a highly optimized CpG cassette into the plasmid backbone as well as by an optimized DNA delivery using an advanced electroporation (EP) technology. By integrating the means for agent administration and EP into a single device, this technology enables a simple, one-step procedure that facilitates reproducibility. We found that highly optimized CpG motifs alone triggers an enhanced IFN-γ and granzyme B response in Elispot assays as well as stronger tumor regression. Furthermore, these effects could be dramatically enhanced when the CpG cassette containing plasmid was administered via the newly developed EP technology. These data suggest that an optimized application of CpG-enriched DNA vaccines may be an attractive strategy for the treatment of cancer. Collectively, these results provide a basis for the transfer of preclinical therapeutic DNA-based immunization studies into successful clinical cancer trials.
