B.S., University of California, Santa Cruz, Bioengineering (2012)
Ph.D., Mayo Clinic Graduate School of Biomedical Sciences, Virology and Gene Therapy (2021)
Mark Kay, Postdoctoral Faculty Sponsor
Ex Vivo and In Vivo CD46 Receptor Utilization by Species D Human Adenovirus Serotype 26 (HAdV26).
Journal of virology
2022; 96 (3): e0082621
Human adenovirus serotype 26 (Ad26) is used as a gene-based vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and HIV-1. However, its primary receptor portfolio remains controversial, potentially including sialic acid, coxsackie and adenovirus receptor (CAR), integrins, and CD46. We and others have shown that Ad26 can use CD46, but these observations were questioned on the basis of the inability to cocrystallize Ad26 fiber with CD46. Recent work demonstrated that Ad26 binds CD46 with its hexon protein rather than its fiber. We examined the functional consequences of Ad26 for infection in vitro and in vivo. Ectopic expression of human CD46 on Chinese hamster ovary cells increased Ad26 infection significantly. Deletion of the complement control protein domain CCP1 or CCP2 or the serine-threonine-proline (STP) region of CD46 reduced infection. Comparing wild-type and sialic acid-deficient CHO cells, we show that the usage of CD46 is independent of its sialylation status. Ad26 transduction was increased in CD46 transgenic mice after intramuscular (i.m.) injection but not after intranasal (i.n.) administration. Ad26 transduction was 10-fold lower than Ad5 transduction after intratumoral (i.t.) injection of CD46-expressing tumors. Ad26 transduction of liver was 1,000-fold lower than that ofAd5 after intravenous (i.v.) injection. These data demonstrate the use of CD46 by Ad26 in certain situations but also show that the receptor has little consequence by other routes of administration. Finally, i.v. injection of high doses of Ad26 into CD46 mice induced release of liver enzymes into the bloodstream and reduced white blood cell counts but did not induce thrombocytopenia. This suggests that Ad26 virions do not induce direct clotting side effects seen during coronavirus disease 2019 (COVID-19) vaccination with this serotype of adenovirus. IMPORTANCE The human species D Ad26 is being investigated as a low-seroprevalence vector for oncolytic virotherapy and gene-based vaccination against HIV-1 and SARS-CoV-2. However, there is debate in the literature about its tropism and receptor utilization, which directly influence its efficiency for certain applications. This work was aimed at determining which receptor(s) this virus uses for infection and its role in virus biology, vaccine efficacy, and, importantly, vaccine safety.
View details for DOI 10.1128/JVI.00826-21
View details for PubMedID 34787457
View details for PubMedCentralID PMC8826919
Unlocking loxP to Track Genome Editing In Vivo.
2021; 12 (8)
The development of CRISPR-associated proteins, such as Cas9, has led to increased accessibility and ease of use in genome editing. However, additional tools are needed to quantify and identify successful genome editing events in living animals. We developed a method to rapidly quantify and monitor gene editing activity non-invasively in living animals that also facilitates confocal microscopy and nucleotide level analyses. Here we report a new CRISPR "fingerprinting" approach to activating luciferase and fluorescent proteins in mice as a function of gene editing. This system is based on experience with our prior cre recombinase (cre)-detector system and is designed for Cas editors able to target loxP including gRNAs for SaCas9 and ErCas12a. These CRISPRs cut specifically within loxP, an approach that is a departure from previous gene editing in vivo activity detection techniques that targeted adjacent stop sequences. In this sensor paradigm, CRISPR activity was monitored non-invasively in living cre reporter mice (FVB.129S6(B6)-Gt(ROSA)26Sortm1(Luc)Kael/J and Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J, which will be referred to as LSL-luciferase and mT/mG throughout the paper) after intramuscular or intravenous hydrodynamic plasmid injections, demonstrating utility in two diverse organ systems. The same genome-editing event was examined at the cellular level in specific tissues by confocal microscopy to determine the identity and frequency of successfully genome-edited cells. Further, SaCas9 induced targeted editing at efficiencies that were comparable to cre, demonstrating high effective delivery and activity in a whole animal. This work establishes genome editing tools and models to track CRISPR editing in vivo non-invasively and to fingerprint the identity of targeted cells. This approach also enables similar utility for any of the thousands of previously generated loxP animal models.
View details for DOI 10.3390/genes12081204
View details for PubMedID 34440379
View details for PubMedCentralID PMC8394901
Improving Molecular Therapy in the Kidney.
Molecular diagnosis & therapy
2020; 24 (4): 375-396
Mutations in approximately 80 genes have been implicated as the cause of various genetic kidney diseases. However, gene delivery to kidney cells from the blood is inefficient because of the natural filtering functions of the glomerulus, and research into and development of gene therapy directed toward kidney disease has lagged behind as compared with hepatic, neuromuscular, and ocular gene therapy. This lack of progress is in spite of numerous genetic mouse models of human disease available to the research community and many vectors in existence that can theoretically deliver genes to kidney cells with high efficiency. In the past decade, several groups have begun to develop novel injection techniques in mice, such as retrograde ureter, renal vein, and direct subcapsular injections to help resolve the issue of gene delivery to the kidney through the blood. In addition, the ability to retarget vectors specifically toward kidney cells has been underutilized but shows promise. This review discusses how recent advances in gene delivery to the kidney and the field of gene therapy can leverage the wealth of knowledge of kidney genetics to work toward developing gene therapy products for patients with kidney disease.
View details for DOI 10.1007/s40291-020-00467-6
View details for PubMedID 32323260
View details for PubMedCentralID PMC7367759
Retargeting adenoviruses for therapeutic applications and vaccines.
2020; 594 (12): 1918-1946
Adenoviruses (Ads) are robust vectors for therapeutic applications and vaccines, but their use can be limited by differences in their in vitro and in vivo pharmacologies. This review emphasizes that there is not just one Ad, but a whole virome of diverse viruses that can be used as therapeutics. It discusses that true vector targeting involves not only retargeting viruses, but importantly also detargeting the viruses from off-target cells.
View details for DOI 10.1002/1873-3468.13731
View details for PubMedID 31944286
View details for PubMedCentralID PMC7311308
Comparison of Gene Delivery to the Kidney by Adenovirus, Adeno-Associated Virus, and Lentiviral Vectors After Intravenous and Direct Kidney Injections.
Human gene therapy
2019; 30 (12): 1559-1571
There are many kidney diseases that might be addressed by gene therapy. However, gene delivery to kidney cells is inefficient. This is due, in part, to the fact that the kidney excludes molecules above 50 kDa and that most gene delivery vectors are megaDaltons in mass. We compared the ability of adeno-associated virus (AAV), adenovirus (Ad), and lentiviral (LV) vectors to deliver genes to renal cells. When vectors were delivered by the intravenous (IV) route in mice, weak luciferase activity was observed in the kidney with substantially more in the liver. When gene delivery was observed in the kidney, expression was primarily in the glomerulus. To avoid these limitations, vectors were injected directly into the kidney by retrograde ureteral (RU) and subcapsular (SC) injections in mice. Small AAV vectors transduced the kidney, but also leaked from the organ and mediated higher levels of transduction in off-target tissues. Comparison of AAV2, 6.2, 8, and rh10 vectors by direct kidney injection demonstrated highest delivery by AAV6.2 and 8. Larger Ad and LV vectors transduced kidney cells and mediated less off-target tissue transduction. These data demonstrate the utility of direct kidney injections to circumvent the kidney size exclusion barrier. They also identify the effects of vector size on on-target and off-target transduction. This lays the foundation for the use of different vector platforms for gene therapy of diverse kidney diseases.
View details for DOI 10.1089/hum.2019.127
View details for PubMedID 31637925
View details for PubMedCentralID PMC6919283
Whole genome comparison of a large collection of mycobacteriophages reveals a continuum of phage genetic diversity.
2015; 4: e06416
The bacteriophage population is large, dynamic, ancient, and genetically diverse. Limited genomic information shows that phage genomes are mosaic, and the genetic architecture of phage populations remains ill-defined. To understand the population structure of phages infecting a single host strain, we isolated, sequenced, and compared 627 phages of Mycobacterium smegmatis. Their genetic diversity is considerable, and there are 28 distinct genomic types (clusters) with related nucleotide sequences. However, amino acid sequence comparisons show pervasive genomic mosaicism, and quantification of inter-cluster and intra-cluster relatedness reveals a continuum of genetic diversity, albeit with uneven representation of different phages. Furthermore, rarefaction analysis shows that the mycobacteriophage population is not closed, and there is a constant influx of genes from other sources. Phage isolation and analysis was performed by a large consortium of academic institutions, illustrating the substantial benefits of a disseminated, structured program involving large numbers of freshman undergraduates in scientific discovery.
View details for DOI 10.7554/eLife.06416
View details for PubMedID 25919952
View details for PubMedCentralID PMC4408529
Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution.
2011; 6 (1): e16329
Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists.
View details for DOI 10.1371/journal.pone.0016329
View details for PubMedID 21298013
View details for PubMedCentralID PMC3029335