Browsing by Subject "Transduction"

Now showing 1 - 3 of 3
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    A novel class of self-complementary AAV vectors with multiple advantages based on cceAAV lacking mutant ITR
    (Elsevier, 2024-02-03) Zhang, Junping; Frabutt, Dylan A.; Chrzanowski, Matthew; Li, Ning; Miller, Lohra M.; Tian, Jiahe; Mulcrone, Patrick L.; Lam, Anh K.; Draper, Benjamin E.; Jarrold, Martin F.; Herzog, Roland W.; Xiao, Weidong; Pediatrics, School of Medicine
    Self-complementary AAV vectors (scAAV) use a mutant inverted terminal repeat (mITR) for efficient packaging of complementary stranded DNA, enabling rapid transgene expression. However, inefficient resolution at the mITR leads to the packaging of monomeric or subgenomic AAV genomes. These noncanonical particles reduce transgene expression and may affect the safety of gene transfer. To address these issues, we have developed a novel class of scAAV vectors called covalently closed-end double-stranded AAV (cceAAV) that eliminate the mITR resolution step during production. Instead of using a mutant ITR, we used a 56-bp recognition sequence of protelomerase (TelN) to covalently join the top and bottom strands, allowing the vector to be generated with just a single ITR. To produce cceAAV vectors, the vector plasmid is initially digested with TelN, purified, and then subjected to a standard triple-plasmid transfection protocol followed by traditional AAV vector purification procedures. Such cceAAV vectors demonstrate yields comparable to scAAV vectors. Notably, we observed enhanced transgene expression as compared to traditional scAAV vectors. The treatment of mice with hemophilia B with cceAAV-FIX resulted in significantly enhanced long-term FIX expression. The cceAAV vectors hold several advantages over scAAV vectors, potentially leading to the development of improved human gene therapy drugs.
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    Comprehensive Comparison of AAV Purification Methods: Iodixanol Gradient Centrifugation vs. Immuno-Affinity Chromatography
    (Hindawi, 2023) Lam, Anh K.; Mulcrone, Patrick L.; Frabutt, Dylan; Zhang, Junping; Chrzanowski, Matthew; Arisa, Sreevani; Munoz, Maite; Li, Xin; Biswas, Moanaro; Markusic, David; Herzog, Roland W.; Xiao, Weidong; Pediatrics, School of Medicine
    Recombinant adeno-associated viruses (AAVs) have emerged as a widely used gene delivery platform for both basic research and human gene therapy. To ensure and improve the safety profile of AAV vectors, substantial efforts have been dedicated to the vector production process development using suspension HEK293 cells. Here, we studied and compared two downstream purification methods, iodixanol gradient ultracentrifugation versus immuno-affinity chromatography (POROS™ CaptureSelect™ AAVX column). We tested multiple vector batches that were separately produced (including AAV5, AAV8, and AAV9 serotypes). To account for batch-to-batch variability, each batch was halved for subsequent purification by either iodixanol gradient centrifugation or affinity chromatography. In parallel, purified vectors were characterized, and transduction was compared both in vitro and in vivo in mice (using multiple transgenes: Gaussia luciferase, eGFP, and human factor IX). Each purification method was found to have its own advantages and disadvantages regarding purity, viral genome (vg) recovery, and relative empty particle content. Differences in transduction efficiency were found to reflect batch-to-batch variability rather than disparities between the two purification methods, which were similarly capable of yielding potent AAV vectors.
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    Understanding mechanical environment changes and biological responses to canine retraction using t-loop
    (2015-05) Jiang, Feifei; Chen, Jie; Bajaj, Anil; El-Mounayri, Hazim; Katona, Thomas; Nauman, Eric
    Predictability of tooth displacement in response to specific orthodontic load system directly links to the quality and effectiveness of the treatment. The key questions are how the tooth’s environment changes in response to the orthodontic load and how the biological tissues respond clinically. The objectives of this study are to determine the mechanical environment (ME) changes and to quantify the biological tissues’ response. Eighteen (18) patients who needed maxillary bilateral canine retractions were involved in the study. A method was developed to quantify the 3D load systems on the canine, which allowed the treatment strategies to be customized in terms of orthodontic loading systems to meet either translation (TR) or controlled tipping (CT) requirement. Dental casts were made before and after each treatment interval, and the Cone Beam Computed Tomography (CBCT) scans were taken prior to and following the entire treatment for control of treatment strategy and post treatment evaluations. Finite element method (FEM) was applied to calculate the location of center of resistance (CRes) for tooth movement control. The location and variation of CRes were recorded and compared with previous studies. A quick CRes assessment method that locates CRes by calculating the centroid of the contact surface (CCS) and the centroid of the projection of root surface (CPCS) in certain direction was also tested and compared with the results from FEM. Customized T-loop spring, a kind of orthodontic appliance, was designed, fabricated, and calibrated on a load measuring system to ensure that the load met the clinician’s prescription. The treatment outcomes in terms of tooth displacement and root resorption characterized by the changes of tooth length and volume as well as the bone mineral density (BMD) represented by the Hounsfield units (HU) change were recorded and analyzed. The ME in terms of stress were also calculated by using FEM. Paired t-test and mixed model ANOVA methods were used to analyze the relationships between the mechanical inputs (quantified and customized load, and corresponding stress) and clinical outcomes (root resorption and BMD change). It was found that the overall root resorption is not significant for canine retraction, but apical root resorption does occur, meaning that orthodontic load is not a sufficient factor. Also, it was observed that HU distribution changed significantly in both root and alveolar bone. The maximum reduction was on the coronal level in the direction perpendicular to the direction of movement in root, and in the direction of the tooth movement at the coronal level in bone. In addition, it was determined that the locations of the CRes in the MD and BL directions were significantly different. The locations of the CRes of a human canine in MD and BL directions can be estimated by finding the CPCSs in the two directions. Finally, it was shown that the stress invariants can be used to characterize how the osteocytes feel when ME changes. The stress invariants in the alveolar bone are not significantly affected by different M/F. The higher bone modeling/remodeling activities along the direction of tooth movement may be related to the initial volumetric increase and decrease in the alveolar bone.