IU Indianapolis ScholarWorks :: Browsing by Author "Lam, Anh K."

Browsing by Author "Lam, Anh K."

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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.
B cell focused transient immune suppression protocol for efficient AAV readministration to the liver
(Elsevier, 2024-02-20) Rana, Jyoti; Herzog, Roland W.; Muñoz-Melero, Maite; Yamada, Kentaro; Kumar, Sandeep R. P.; Lam, Anh K.; Markusic, David M.; Duan, Dongsheng; Terhorst, Cox; Byrne, Barry J.; Corti, Manuela; Biswas, Moanaro; Pediatrics, School of Medicine
Adeno-associated virus (AAV) vectors are used for correcting multiple genetic disorders. Although the goal is to achieve lifelong correction with a single vector administration, the ability to redose would enable the extension of therapy in cases in which initial gene transfer is insufficient to achieve a lasting cure, episomal vector forms are lost in growing organs of pediatric patients, or transgene expression is diminished over time. However, AAV typically induces potent and long-lasting neutralizing antibodies (NAbs) against capsid that prevents re-administration. To prevent NAb formation in hepatic AAV8 gene transfer, we developed a transient B cell-targeting protocol using a combination of monoclonal Ab therapy against CD20 (for B cell depletion) and BAFF (to slow B cell repopulation). Initiation of immunosuppression before (rather than at the time of) vector administration and prolonged anti-BAFF treatment prevented immune responses against the transgene product and abrogated prolonged IgM formation. As a result, vector re-administration after immune reconstitution was highly effective. Interestingly, re-administration before the immune system had fully recovered achieved further elevated levels of transgene expression. Finally, this immunosuppression protocol reduced Ig-mediated AAV uptake by immune cell types with implications to reduce the risk of immunotoxicities in human gene therapy with AAV.
Chemical modification of AAV9 capsid with N-ethyl maleimide alters vector tissue tropism
(Springer Nature, 2023-05-25) Mulcrone, Patrick L.; Lam, Anh K.; Frabutt, Dylan; Zhang, Junping; Chrzanowski, Matthew; Herzog, Roland W.; Xiao, Weidon; Pediatrics, School of Medicine
Although more adeno-associated virus AAV-based drugs enter the clinic, vector tissue tropism remains an unresolved challenge that limits its full potential despite that the tissue tropism of naturally occurring AAV serotypes can be altered by genetic engineering capsid vie DNA shuffling, or molecular evolution. To further expand the tropism and thus potential applications of AAV vectors, we utilized an alternative approach that employs chemical modifications to covalently link small molecules to reactive exposed Lysine residues of AAV capsids. We demonstrated that AAV9 capsid modified with N-ethyl Maleimide (NEM) increased its tropism more towards murine bone marrow (osteoblast lineage) while decreased transduction of liver tissue compared to the unmodified capsid. In the bone marrow, AAV9-NEM transduced Cd31, Cd34, and Cd90 expressing cells at a higher percentage than unmodified AAV9. Moreover, AAV9-NEM localized strongly in vivo to cells lining the calcified trabecular bone and transduced primary murine osteoblasts in culture, while WT AAV9 transduced undifferentiated bone marrow stromal cells as well as osteoblasts. Our approach could provide a promising platform for expanding clinical AAV development to treat bone pathologies such as cancer and osteoporosis. Thus, chemical engineering the AAV capsid holds great potential for development of future generations of AAV vectors.
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.
Cryptic resolution sites in the vector plasmid lead to the heterogeneities in the rAAV vectors
(Wiley, 2023) Zhang, Junping; Chrzanowski, Matthew; Frabutt, Dylan A.; Lam, Anh K.; Mulcrone, Patrick L.; Li, Lei; Konkle, Barbara A.; Miao, Carol H.; Xiao, Weidong; Pediatrics, School of Medicine
Recombinant adeno-associated virus (rAAV) vectors carry a cassette of interest retaining only the inverted terminal repeats (ITRs) from the wild-type virus. Conventional rAAV production primarily uses a vector plasmid as well as helper genes essential for AAV replication and packaging. Nevertheless, plasmid backbone related contaminants have been a major source of vector heterogeneity. The mechanism driving the contamination phenomenon has yet to be elucidated. Here we identified cryptic resolution sites in the plasmid backbone as a key source for producing snapback genomes, which leads to the increase of vector genome heterogeneity in encapsidated virions. By using a single ITR plasmid as a model molecule and mapping subgenomic particles, we found that there exist a few typical DNA break hotspots in the vector DNA plasmid backbone, for example, on the ampicillin DNA element, called aberrant rescue sites. DNA around these specific breakage sites may assume some typical secondary structures. Similar to normal AAV vectors, plasmid DNA with a single ITR was able to rescue and replicate efficiently. These subgenomic DNA species significantly compete for trans factors required for rAAV rescue, replication, and packaging. The replication of single ITR contaminants during AAV production is independent of size. Packaging of these species is greatly affected by its size. A single ITR and a cryptic resolution site in the plasmid work synergistically, likely causing a source of plasmid backbone contamination.
Fast and high-throughput LC-MS characterization, and peptide mapping of engineered AAV capsids using LC-MS/MS
(Elsevier, 2022-09-24) Lam, Anh K.; Zhang, Junping; Frabutt, Dylan; Mulcrone, Patrick L.; Li, Lei; Zeng, Lifan; Herzog, Roland W.; Xiao, Weidong; Pediatrics, School of Medicine
Adeno-associated virus (AAV) has emerged as a leading platform for gene therapy. With the skyrocketing rate of AAV research and the prevalence of many new engineered capsids being investigated in preclinical and clinical trials, capsid characterization plays a vital role in serotype confirmation and quality control. Further, peptide mapping the capsid proteins might inevitably be a future requirement by regulatory agencies since it is a critical step in good manufacturing practice (GMP) for biotherapeutic characterization. To overcome many challenges that traditional methods like SDS-PAGE and western blots carry, liquid chromatography and mass spectrometry (LC-MS) allows high resolution and sensitivity with great accuracy in characterizing the AAV capsid proteins. Our optimized LC-MS method provides quick sample preparation, a fast and high-throughput 4-min run, and high sensitivity, which allows for very efficient characterization of wild-type and engineered capsids. This study also reports the usage of LC-MS/MS peptide mapping of AAV capsid proteins to determine the most accessible lysine residues targeted by chemical modifications. Our detailed protocols are anticipated to promote the development and discovery of AAV variants with high accuracy and efficiency.
Genomic Designs of rAAVs Contribute to Pathological Changes in the Livers and Spleens of Mice
(Wiley, 2022) Mulcrone, Patrick L.; Zhang, Junping; Pride, P. Melanie; Lam, Anh K.; Frabutt, Dylan A.; Ball-Kell, Susan M.; Xiao, Weidong; Pediatrics, School of Medicine
Recombinant AAV (rAAV) gene therapy is being investigated as an effective therapy for several diseases including hemophilia B. Reports of liver tumor development in certain mouse models due to AAV treatment and genomic integration of the rAAV vector has raised concerns about the long-term safety and efficacy of this gene therapy. To investigate whether rAAV treatment causes cancer, we utilized two mouse models, inbred C57BL/6 and hemophilia B Balb/C mice (HemB), to test if injecting a high dose of various rAAV8 vectors containing or lacking hFIX transgene, a Poly-A sequence, or the CB or TTR promoter triggered liver fibrosis and/or cancer development over the course of the 6.5-month study. We observed no liver tumors in either mouse cohort regardless of rAAV treatment through ultrasound imaging, gross anatomical assessment at sacrifice, and histology. We did, however, detect differences in collagen deposition in C57BL/6 livers and HemB spleens of rAAV-injected mice. Pathology reports of the HemB mice revealed many pathological phenomena, including fibrosis and inflammation in the livers and spleens across different AAV-injected HemB mice. Mice from both cohorts injected with the TTR-hFIX vector demonstrated minimal adverse events. While not tumorigenic, high dose of rAAVs, especially those with incomplete genomes, can influence liver and spleen health negatively that could be problematic for cementing AAVs as a broad therapeutic option in the clinic.
Subgenomic particles in rAAV vectors result from DNA lesion/break and non-homologous end joining of vector genomes
(Elsevier, 2022-08-24) Zhang, Junping; Guo, Ping; Yu, Xiangping; Frabutt, Dylan A.; Lam, Anh K.; Mulcrone, Patrick L.; Chrzanowski, Matthew; Firrman, Jenni; Pouchnik, Derek; Sang, Nianli; Diao, Yong; Herzog, Roland W.; Xiao, Weidong; Pediatrics, School of Medicine
Recombinant adeno-associated virus (rAAV) vectors have been developed for therapeutic treatment of genetic diseases. Current rAAV vectors administered to affected individuals often contain vector DNA-related contaminants. Here we present a thorough molecular analysis of the configuration of non-standard AAV genomes generated during rAAV production using single-molecule sequencing. In addition to the sub-vector genomic-size particles containing incomplete AAV genomes, our results showed that rAAV preparations were contaminated with multiple categories of subgenomic particles with a snapback genome (SBG) configuration or a vector genome with deletions. Through CRISPR and nuclease-based modeling in tissue culture cells, we identified that a potential mechanism leading to formation of non-canonical genome particles occurred through non-homologous end joining of fragmented vector genomes caused by genome lesions or DNA breaks present in the host cells. The results of this study advance our understanding of AAV vectors and provide new clues for improving vector efficiency and safety profiles for use in human gene therapy.
β Cell microRNAs Function as Molecular Hubs of Type 1 Diabetes Pathogenesis and as Biomarkers of Diabetes Risk
(bioRxiv, 2023-06-15) Syed, Farooq; Krishnan, Preethi; Chang, Garrick; Langlais, Sarah R.; Hati, Sumon; Yamada, Kentaro; Lam, Anh K.; Talware, Sayali; Liu, Xiaowen; Sardar, Rajesh; Liu, Jing; Mirmira, Raghavendra G.; Evans-Molina, Carmella; Pediatrics, School of Medicine
MicroRNAs (miRNAs) are small non-coding RNAs that play a crucial role in modulating gene expression and are enriched in cell-derived extracellular vesicles (EVs). We investigated whether miRNAs from human islets and islet-derived EVs could provide insight into β cell stress pathways activated during type 1 diabetes (T1D) evolution, therefore serving as potential disease biomarkers. We treated human islets from 10 cadaveric donors with IL-1β and IFN-γ to model T1D ex vivo. MicroRNAs were isolated from islets and islet-derived EVs, and small RNA sequencing was performed. We found 20 and 14 differentially expressed (DE) miRNAs in cytokine- versus control-treated islets and EVs, respectively. Interestingly, the miRNAs found in EVs were mostly different from those found in islets. Only two miRNAs, miR-155-5p and miR-146a-5p, were upregulated in both islets and EVs, suggesting selective sorting of miRNAs into EVs. We used machine learning algorithms to rank DE EV-associated miRNAs, and developed custom label-free Localized Surface Plasmon Resonance-based biosensors to measure top ranked EVs in human plasma. Results from this analysis revealed that miR-155, miR-146, miR-30c, and miR-802 were upregulated and miR-124-3p was downregulated in plasma-derived EVs from children with recent-onset T1D. In addition, miR-146 and miR-30c were upregulated in plasma-derived EVs of autoantibody positive (AAb+) children compared to matched non-diabetic controls, while miR-124 was downregulated in both T1D and AAb+ groups. Furthermore, single-molecule fluorescence in situ hybridization confirmed increased expression of the most highly upregulated islet miRNA, miR-155, in pancreatic sections from organ donors with AAb+ and T1D.

Browsing by Author "Lam, Anh K."

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