scholarly journals A truncated reverse transcriptase enhances prime editing by split AAV vectors

2021 ◽  
Author(s):  
Zongliang Gao ◽  
Jakob Haldrup ◽  
Sujan Ravendran ◽  
Nanna S Mikkelsen ◽  
Jacob Giehm Mikkelsen ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Reverse Transcriptase ◽  
Viral Vector ◽  
Codon Optimization ◽  
Rnase H ◽  
Genomic Locus ◽  
Guide Rna ◽  
Split Intein ◽  
Efficient Delivery

Prime editing is a new CRISPR-based genome editing technology that relies on the prime editor (PE), a fusion protein of Cas9-nickase and M-MLV reverse transcriptase (RT), and a prime editing guide RNA (pegRNA) that serves both to target PE to the desired genomic locus and to carry the edit to be introduced. Here, we make advancements to the RT moiety to improve prime editing efficiencies and truncations to mitigate issues with AAV viral vector size limitations, which currently do not support efficient delivery of the large prime editing components. These efforts include RT variant screening, codon optimization, and PE truncation by removal of the RNase H domain and further trimming. This led to a codon-optimized and size-minimized PE that has an expression advantage (1.4x fold) and size advantage (621 bp shorter). In addition, we optimize the split intein PE system and identify Rma-based Cas9 split sites (573-574 and 673-674) that combined with the truncated PE delivered by dual AAVs result in superior AAV titer and prime editing efficiency. This novel minimized PE provides great value to AAV-based delivery applications in vivo.

scholarly journals Development of a flexible split prime editor using truncated reverse transcriptase

2021 ◽  
Author(s):  
Wen Xue ◽  
Chunwei Zheng ◽  
SHUNQING LIANG ◽  
Pengpeng Liu ◽  
Bin Liu ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Mammalian Cells ◽  
Stop Codon ◽  
Protein Biosynthesis ◽  
Potential Effect ◽  
Rnase H ◽  
Full Length ◽  
Large Size ◽  
Efficient Delivery

Prime Editor (PE) has tremendous promise for gene therapy. However, it remains a challenge to deliver PE (>6.3 kb) in vivo. Although PE can be split into two fragments and delivered using dual adeno-associated viruses (AAVs), choice of split sites within Cas9, which affects editing efficiency, is limited due to the large size of PE. Furthermore, the potential effect of overexpressing RT in mammalian cells is largely unknown. Here, we developed a compact PE with complete deletion of the RNase H domain of reverse transcriptase (RT), which showed comparable editing to full-length PE. Using compact PE, we tested the effect of 4 different Cas9 split sites and found that the Glu 573 split site supports robust editing (up to 93% of full-length PE). The compact PE, but not PE2, abolished its binding to eRF1 and showed minimal effect on stop codon readthrough, which therefore might reduce the effects on protein biosynthesis. This study identifies a safe and efficient compact PE2 that enables flexible split-PE design to facilitate efficient delivery in vivo and advance the utility of prime editing.

scholarly journals Functionalized lipid-like nanoparticles for in vivo mRNA delivery and base editing

2020 ◽  
Vol 6 (34) ◽  
pp. eabc2315 ◽  
Author(s):  
Xinfu Zhang ◽  
Weiyu Zhao ◽  
Giang N. Nguyen ◽  
Chengxiang Zhang ◽  
Chunxi Zeng ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Low Dose ◽  
Genetic Disorders ◽  
Base Editing ◽  
Guide Rna ◽  
Efficient Delivery ◽  
Human Factor Viii ◽  
Mrna Therapeutics ◽  
Further Development

Messenger RNA (mRNA) therapeutics have been explored to treat various genetic disorders. Lipid-derived nanomaterials are currently one of the most promising biomaterials that mediate effective mRNA delivery. However, efficiency and safety of this nanomaterial-based mRNA delivery remains a challenge for clinical applications. Here, we constructed a series of lipid-like nanomaterials (LLNs), named functionalized TT derivatives (FTT), for mRNA-based therapeutic applications in vivo. After screenings on the materials, we identified FTT5 as a lead material for efficient delivery of long mRNAs, such as human factor VIII (hFVIII) mRNA (~4.5 kb) for expression of hFVIII protein in hemophilia A mice. Moreover, FTT5 LLNs demonstrated high percentage of base editing on PCSK9 in vivo at a low dose of base editor mRNA (~5.5 kb) and single guide RNA. Consequently, FTT nanomaterials merit further development for mRNA-based therapy.

scholarly journals Neutralizing epitope of the Fusion Protein of Respiratory Syncytial Virus Embedded in the HA Molecule of LAIV Virus is not Sufficient to Prevent RS Virus Pulmonary Replication but Ameliorates Lung Pathology following RSV Infection in Mice

2020 ◽  
Vol 14 (1) ◽  
pp. 147-156
Author(s):  
Tatiana Kotomina ◽  
Irina Isakova-Sivak ◽  
Ekaterina Stepanova ◽  
Daria Mezhenskaya ◽  
Victoria Matyushenko ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Fusion Protein ◽  
Viral Vector ◽  
Replication Capacity ◽  
Lung Pathology ◽  
Neutralizing Epitope ◽  
Syncytial Virus ◽  
Chimeric Viruses

Aims: To develop experimental bivalent vaccines against influenza and RSV using a cold-adapted LAIV backbone. Background: Respiratory syncytial virus (RSV) is a causative agent of bronchiolitis and pneumonia in young children, elderly and immunocompromised adults. No vaccine against RSV has been licensed to date for various reasons. One of the promising platforms for designing RSV vaccine is the use of live attenuated influenza vaccine (LAIV) viruses to deliver RSV epitopes to the respiratory mucosa. Objective: To generate recombinant LAIV viruses encoding a neutralizing epitope of the RSV fusion protein and assess their protective potential against both influenza and RSV infections in a mouse model. Methods: Reverse genetics methods were used to rescue recombinant LAIV+HA/RSV viruses expressing chimeric hemagglutinins encoding the RSV-F epitope at its N-terminus using two different flexible linkers. BALB/c mice were intranasally immunized with two doses of the recombinant viruses and then challenged with the influenza virus or RSV. The LAIV viral vector and formalin-inactivated RSV (FI-RSV) were included as control vaccines. Protection was assessed by the reduction of virus pulmonary titers. In addition, RSV-induced lung pathology was evaluated by histopathology studies. Results: Two rescued chimeric LAIV+HA/RSV viruses were identical to the LAIV vector in terms of replication capacity in vitro and in vivo. The RSV-F neutralizing epitope was successfully expressed only if inserted into the HA molecule via G-linker, but not A-linker. Both chimeric viruses induced high influenza-specific antibody levels and fully protected mice against a lethal influenza challenge virus. However, they induced weak anti-RSV antibody responses which did not prevent RS virus replication upon challenge, and only LAIV-HA+G-RSV variant protected mice against RSV-induced lung pathology. Conclusion: Although the designed LAIV-RSV chimeric viruses were unable to neutralize the RS virus pulmonary replication, the LAIV-HA+G-RSV reduced RSV-induced lung pathology and can be considered a promising bivalent vaccine against influenza and RSV infections and warrants its further development.

scholarly journals Mutations in the RNase H domain of HIV-1 reverse transcriptase affect the initiation of DNA synthesis and the specificity of RNase H cleavage in vivo

2002 ◽  
Vol 99 (14) ◽  
pp. 9515-9520 ◽  
Author(s):  
J. G. Julias ◽  
M. J. McWilliams ◽  
S. G. Sarafianos ◽  
E. Arnold ◽  
S. H. Hughes
Keyword(s):  
Reverse Transcriptase ◽  
Dna Synthesis ◽  
Rnase H ◽  
Hiv 1

scholarly journals Development of Resistance to 4’-Ethynyl-2-Fluoro-2’-Deoxyadenosine (EFdA) by WT and Nucleoside Reverse Transcriptase Inhibitor Resistant Human Immunodeficiency Virus Type 1

2020 ◽  
Author(s):  
Maria E. Cilento ◽  
Eleftherios Michailidis ◽  
Tatiana V. Ilina ◽  
Eva Nagy ◽  
Hiroaki Mitsuya ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Resistance Mutation ◽  
Transcriptase Inhibitor ◽  
Rnase H ◽  
Phenotypic Resistance ◽  
Development Of Resistance ◽  
Primary Determinant ◽  
Virus Fitness

4’-ethynyl-2-fluoro-2’-deoxyadenosine (EFdA, MK-8591, islatravir) is a nucleoside reverse transcriptase translocation inhibitor (NRTTI) with exceptional potency against WT and drug-resistant HIV strains. However, HIV resistance to EFdA is not well characterized. We therefore developed resistance to EFdA by serial passages using progressively increasing concentrations of EFdA. The starting virus was either WT or clinically relevant NRTI-resistant viruses K65R, M184V, and D67N/K70R/T215F/K219Q). In all cases, the selected mutations included M184V. Additional mutations in the RT connection domain (R358K and E399K) and one mutation in the RNase H domain (A502V) were noted. Site-specific mutagenesis validated the role for M184V as the primary determinant for resistance to EFdA; none of the connection domain mutations contributed significantly to phenotypic resistance to EFdA. A novel EFdA resistance mutation was also observed in the background of M184V. The A114S/M184V combination of mutations imparted higher resistance to EFdA (~24-fold) than M184V (−8-fold) or A114S (~2-fold) alone. Virus fitness data suggested that A114S affects HIV fitness by itself and in the presence of M184V. This is consistent with biochemical experiments that showed decreases in the enzymatic efficiency (kcat/Km) of WT RT vs. A114S (2.1-fold) and A114S/M184V/502V (6.5-fold), whereas there was no significant effect of A502V on RT or virus fitness. The observed EFdA resistance of M184V by itself and in combination with A114S combined with the strong published in vitro and in vivo data, confirm that EFdA is an excellent candidate as a potential HIV therapeutic.

Development of Human Immunodeficiency Virus Type 1Starting with Wild-Type or Nucleoside Reverse Transcriptase Inhibitor Resistant-Strains

2021 ◽  
Author(s):  
Maria E. Cilento ◽  
Aaron B. Reeve ◽  
Eleftherios Michailidis ◽  
Tatiana V. Ilina ◽  
Eva Nagy ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Transcriptase Inhibitor ◽  
Rnase H ◽  
Phase Iii ◽  
Wild Type ◽  
Resistance Mutations ◽  
Primary Determinant ◽  
Hiv 1

4’-ethynyl-2-fluoro-2’-deoxyadenosine (EFdA, MK-8591, islatravir) is a nucleoside reverse transcriptase translocation inhibitor (NRTTI) with exceptional potency against WT and drug-resistant HIV-1, in Phase III clinical trials. EFdA resistance is not well characterized. To study EFdA-resistance patterns as it may emerge in naïve or tenofovir- (TFV), emtricitabine/lamivudine- (FTC/3TC), or zidovudine- (AZT) treated patients we performed viral passaging experiments starting with wild-type, K65R, M184V, or D67N/K70R/T215F/K219Q HIV-1. Regardless the starting viral sequence, all selected EFdA-resistant variants included the M184V RT mutation. Using recombinant viruses, we validated the role for M184V as the primary determinant of EFdA resistance; none of the observed connection subdomain (R358K and E399K) or RNase H domain (A502V) mutations significantly contributed to EFdA resistance. A novel EFdA resistance mutational pattern that included A114S was identified in the background of M184V. A114S/M184V exhibited higher EFdA resistance (∼24-fold) than M184V (∼8-fold) or A114S alone (∼2-fold). Remarkably, A114S/M184V and A114S/M184V/A502V resistance mutations were up to 50-fold more sensitive to tenofovir than WT HIV-1. These mutants also had significantly lower specific infectivity than WT. Biochemical experiments confirmed decreases in the enzymatic efficiency (k cat /K m ) of WT vs. A114S (2.1-fold) and A114S/M184V/A502V (6.5-fold) RTs, with no effect of A502V on enzymatic efficiency or specific infectivity. The rather modest EFdA resistance of M184V or A114S/M184V (8- and 24-fold), their hypersusceptibility to tenofovir, and strong published in vitro and in vivo data, suggest that EFdA is an excellent therapeutic candidate for naïve, AZT-, FTC/3TC, and especially tenofovir-treated patients.

scholarly journals In Vivo Ty1 Reverse Transcription Can Generate Replication Intermediates with Untidy Ends

1998 ◽  
Vol 72 (8) ◽  
pp. 6490-6503 ◽  
Author(s):  
Emilie H. Mules ◽  
Ozcan Uzun ◽  
Abram Gabriel
Keyword(s):  
Reverse Transcriptase ◽  
Reverse Transcription ◽  
Rnase H ◽  
Reverse Transcriptases ◽  
Trna Primer ◽  
Ty1 Retrotransposition ◽  
Retroviral Replication ◽  
Dna Template ◽  
Replication Intermediates

ABSTRACT Ty1 retrotransposition, like retroviral replication, is a complex series of events requiring reverse transcription of an RNA intermediate, RNA-primed minus- and plus-strand DNA synthesis, multiple strand transfers, and precise cleavages of the template and primers by RNase H. In this report, we examine the structure of in vivo Ty1 replication intermediates, specifically with regard to the behavior of reverse transcriptase upon reaching template ends and to the precision with which RNase H might generate these ends. While the expected 3′ termini were always identified, terminal nontemplated bases were also observed at all of the RNA and DNA template ends examined. Nontemplated A residues were most common at all 3′ ends, although C residues were preferentially added to minus-strand termini paused at the 5′ end of capped Ty1 RNA. In addition, we observed that RNase H removal of the tRNA primer and of the polypurine tract was not always precise or efficient. Finally, we noted numerous instances of Ty1 reverse transcriptase transferring from normal Ty1 template ends to various tRNA templates, with continued synthesis to specific modified bases. A similar pattern was obtained for Ty2, indicating that template ends offer unique opportunities for these two related reverse transcriptases to generate errors.

scholarly journals Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase–RNase H domain exhibits polymerase and RNase H activities

2000 ◽  
Vol 348 (2) ◽  
pp. 337-342 ◽  
Author(s):  
Marcelle WILHELM ◽  
Mansour BOUTABOUT ◽  
François-Xavier WILHELM
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Fusion Protein ◽  
Reverse Transcriptase ◽  
Active Sites ◽  
Metal Chelate ◽  
Active Form ◽  
Biochemical Properties ◽  
Rnase H ◽  
Integrase Gene

Replication of the Saccharomyces cerevisiae Ty1 retrotransposon requires a reverse transcriptase capable of synthesizing Ty1 DNA. The first description of an active form of a recombinant Ty1 enzyme with polymerase and RNase H activities is reported here. The Ty1 enzyme was expressed as a hexahistidine-tagged fusion protein in Escherichia coli to facilitate purification of the recombinant protein by metal-chelate chromatography. Catalytic activity of the recombinant protein was detected only when amino acid residues encoded by the integrase gene were added to the N-terminus of the reverse transcriptase-RNase H domain. This suggests that the integrase domain could play a role in proper folding of reverse transcriptase. Several biochemical properties of the Ty1 enzyme were analysed, including the effect of MgCl2, NaCl, temperature and of the chain terminator dideoxy GTP on its polymerase activity. RNase H activity was examined by monitoring the cleavage of a RNA-DNA template-primer. Our results suggest that the distance between the RNase H and polymerase active sites corresponds to the length of a 14-nucleotide RNA-DNA heteroduplex. The recombinant protein produced in E. coli should be useful for further biochemical and structural analyses and for a better understanding of the role of integrase in the activation of reverse transcriptase.

Enhanced Stability and Controlled Delivery of MOF Encapsulated Vaccines and Their Immunogenic Response In Vivo

2018 ◽  
Author(s):  
Michael Luzuriaga ◽  
Raymond P. Welch ◽  
Madushani Dharmawardana ◽  
Candace Benjamin ◽  
Shaobo Li ◽  
...  
Keyword(s):  
Viral Vector ◽  
Controlled Delivery ◽  
Conformational Structure ◽  
Spectroscopy Analysis ◽  
Zeolitic Imidazolate Framework ◽  
Structural Conformation ◽  
Depth Study ◽  
Viral Nanoparticle

<div><div><div><p>Vaccines have an innate tendency to lose their structural conformation upon environmental and chemical stressors. A loss in conformation reduces the therapeutic ability to prevent the spread of a pathogen. Herein, we report an in-depth study of zeolitic imidazolate framework-8 (ZIF-8) and its ability to provide protection for a model viral vector against dena- turing conditions. The immunoassay and spectroscopy analysis together demonstrate enhanced thermal and chemical stability to the conformational structure of the encapsulated viral nanoparticle. The long-term biological activity of this virus-ZIF composite was investigated in animal models to further elucidate the integrity of the encapsulated virus, the bio-safety, and immunogenicity of the overall composite. Additionally, histological analysis found no observable tissue damage in the skin or vital organs in mice, following multiple subcutaneous administrations. This study shows that ZIF-based protein composites are strong candidates for improved preservation of proteinaceous drugs, are biocompatible, and capable of controlling the release and adsorption of drugs in vivo.</p></div></div></div>

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