Biology subjects

Mohler, P. J.

Publications and source records attributed to Mohler, P. J..

3 recordsLinked to original sources

bioRxiv2022

Differential Evasion of Delta and Omicron Immunity and Enhanced Fusogenicity of SARS-CoV-2 Omicron BA.4/5 and BA.2.12.1 Subvariants

The rising case numbers of the SARS-CoV-2 Omicron BA.4, BA.5, and BA.2.12.1 subvariants has generated serious concern about the course of the pandemic. Here we examine the neutralization resistance, infectivity, processing, and fusogenicity of spike from the BA.4/5 and BA.2.12.1 SARS-CoV-2 variants compared with other Omicron subvariants and Delta. Critically, we found that the new Omicron subvariants BA.4/5 and BA.2.12.1 were more resistant to neutralization by mRNA-vaccinated and boosted health care worker sera and Omicron-BA.1-wave patient sera than were the BA.1 and BA.2 variants. Interestingly, Delta-wave patient sera neutralized more efficiently against not only Delta but also BA.4/5 and BA.2.12.1 variants that also contain substitutions at position L452, similar to Delta. The BA.4/5 and BA.2.12.1 variants also exhibited higher fusogenicity, and increased spike processing, dependent on the L452 substitution. These results highlight the key role of the L452R and L452Q mutations in BA.4/5 and BA.2.12.1 subvariants.

microbiology↗

bioRxiv2020

Efficient precise in vivo base editing in adult dystrophic mice

ABSTRACTBackground Recent advances in the base editing technology have created an exciting opportunity to precisely correct disease-causing mutations. However, the large size of base editors and their inherited off-target activities pose challenges for in vivo base editing. Moreover, the requirement of a protospacer adjacent motif (PAM) sequence within a suitable window near the mutation site further limits the targeting feasibility. In this work, we rationally improved the adenine base editor (ABE) to overcome these challenges and demonstrated the exceptionally high efficiency to precisely edit the Duchenne muscular dystrophy (DMD) mutation in adult mice.Methods We employed a fluorescence reporter assay to assess the feasibility of ABE to correct the dystrophin mutation in mdx4cv mice. The intein protein trans-splicing (PTS) was used to split the oversized ABE into two halves for efficient packaging into adeno-associated virus 9 (AAV9). The ABE with broadened PAM recognition (ABE-NG) was rationally re-designed for improved off-target RNA editing activity and on-target DNA editing efficiency. The mdx4cv mice at the 5 weeks of age receiving intramuscular or intravenous injections of AAV9 carrying the improved ABE-NG were analyzed at 10 weeks or 10 months of age. The editing outcomes were analyzed by Sanger and deep sequencing of the amplicons, immunofluorescence staining, Western blot and contractile function measurements. The off-target activities, host immune response and long-term toxicity were analyzed by deep sequencing, ELISA and serological assays, respectively.Results We showed efficient in vitro base correction of the dystrophin mutation carried in mdx4cv mice using ABE-NG. The super-fast intein-splits of ABE-NG enabled the expression of full-length ABE-NG and efficient AAV9 packaging. We rationally improved ABE-NG with eliminated off-target RNA editing activity and minimal PAM requirement, and packaged into AAV9 (AAV9-iNG). Intramuscular and intravenous administration of AAV9-iNG resulted in dystrophin restoration and functional improvement. At 10 months after AAV9-iNG treatment, a near complete rescue of dystrophin was measured in mdx4cv mouse hearts. The off-target activities remained low and no obvious toxicity was detected.Conclusions This study highlights the promise of permanent base editing using iABE-NG for the treatment of monogenic diseases, in particular, the genetic cardiomyopathies.Competing Interest StatementThe authors have submitted a patent application based on the results reported in this paper.View Full Text

bioengineering↗

bioRxiv2019

Overexpression of a non-muscle RBFOX2 isoform triggers cardiac conduction defects in myotonic dystrophy

Myotonic dystrophy type 1 (DM1) is a multisystemic genetic disorder caused by a CTG trinucleotide repeat expansion in the 3' untranslated region of DMPK gene. Heart dysfunctions occur in nearly 80% of DM1 patients and are the second leading cause of DM1-related deaths. Despite these figures, the mechanisms underlying cardiac-based DM1 phenotypes are unknown. Herein, we report that upregulation of a non-muscle splice isoform of RNA binding protein RBFOX2 in DM1 heart tissue--due to altered splicing factor and microRNA activities--induces cardiac conduction defects in DM1 individuals. Mice engineered to express the non-muscle RBFOX2 isoform in heart via tetracycline-inducible transgenesis, or CRISPR/Cas9-mediated genome editing, reproduced DM1-related cardiac-conduction delay and spontaneous episodes of arrhythmia. Further, by integrating RNA binding with cardiac transcriptome datasets from both DM1 patients and mice expressing the non-muscle RBFOX2 isoform, we identified RBFOX2-driven splicing defects in the voltage-gated sodium and potassium channels, which can alter their electrophysiological properties. Thus, our results uncover a trans-dominant role for an aberrantly expressed RBFOX2 isoform in DM1 cardiac pathogenesis.

genetics↗