Hosseinali, Mehraveh2026-01-302026-01-302026-01-302026-01-30https://hdl.handle.net/10012/22918Gene therapy holds significant promise for treating various diseases, with Adeno-associated virus (AAV) vectors being among the most widely used delivery systems. However, current standard AAV production methods relying on costly and inefficient mammalian cell culture limit scalability and clinical accessibility. A similar human virus, Torque teno virus (TTV), also holds great potential for gene therapy; however, it also suffers from problems in its production. To address this manufacturing bottleneck, this study aimed to develop a novel, cost-effective platform for hybrid viral vector production entirely within Escherichia coli. This work advances research on the use of miniphagemids, phages that package a minimal vector genome, to achieve in-bacterial assembly of novel hybrid AAV serotype 2 (AAV2)-based and TTV genotype 19 (TTV19)-based vectors. The hypothesis being tested is that the co-production of single stranded DNA using miniphagemid technology and key AAV or TTV proteins in Escherichia coli can result in AAV-based or TTV-based vectors. The key objectives were therefore: 1) showing recombinant expression of heterologous capsid proteins AAV2 VP1/VP2/VP3 and TTV19 ORF1 in E. coli; 2) producing ssDNA minigenomes flanked by either AAV2 inverted terminal repeat (ITR) or TTV19 untranslated terminal repeat (UTR) sequences; and 3) showing that co-producing protein(s) and ssDNA results in AAV2- or TTV19-based vectors. Results confirmed that AAV2 VP2 and VP3 could be produced in E. coli, albeit expressed primarily as insoluble inclusion bodies. Transformation of cells with a plasmid encoding VP1 resulted in reduced growth and no VP1 was recovered. Expression of TTV19 ORF1 in E. coli produced two histidine-tagged protein products approximately half the size of the expected protein (as previously reported). The ssDNA minigenomes were successfully produced and purified, exhibiting high purity (Objective 2). The central finding was the successful in-bacterial production and purification of functional hybrid vectors, termed AAV-based and TTV-based (Objective 3). Iodixanol gradient ultracentrifugation confirmed particle assembly and density separation. Subsequent qPCR quantification demonstrated high genomic titers in the purified fractions, providing strong evidence of successful ssDNA encapsulation by the heterologous capsids within the E. coli host. The study further found that the TTV19 UTRs likely enhance packaging efficiency in the TTV-based hybrid vector system. In conclusion, this research establishes a robust and scalable E. coli-based platform for producing non-enveloped hybrid viral vectors. This achievement represents a significant step toward revolutionizing gene therapy vector manufacturing, offering a pathway to highly purified, consistent, and affordable therapeutic vectors.enViral Gene TherapyAdeno-Associated VirusM13 MiniphagemidTorque Teno VirusHybrid Gene Therapy VectorMaster Thesis