Gene editing methods and compositions for eliminating risk of JC virus activation and PML (progressive multifocal leukoencephalopathy) during immunosuppressive therapy
Inventors
Khalili, Kamel • Malcolm, Thomas • Kohn, Kenneth I.
Assignees
Temple Univ School of Medicine • Excision BioTherapeutics Inc
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Publication Number
US-10279014-B2
Publication Date
2019-05-07
Expiration Date
Abstract
A method of eliminating the risk of JCV activation in a subject undergoing immunosuppressive therapy, by administering an effective amount of a gene editing composition directed toward at least one target sequence in the JCV genome, cleaving the target sequence in the JCV genome, disrupting the JCV genome, eliminating the JCV infection, eliminating the risk of JCV activation, and treating the subject with an immunosuppressive therapy. A pharmaceutical composition including at least one isolated nucleic acid sequence encoding a CRISPR-associated endonuclease and at least one gRNA having a spacer sequence complementary to a target sequence in a JCV DNA, the isolated nucleic acid sequences being included in at least one expression vector. Pharmaceutical compositions including at least one isolated nucleic acid sequence encoding at least one TALEN, at least one ZFN, and gene editing composition of C2c1, C2c3, TevCas9, Archaea Cas9, CasY.1-CasY.6, CasX, or argonaute protein, which target at least one nucleotide sequence of the JCV genome.
Core Innovation
The invention relates to a gene editing approach for a subject latently infected with John Cunningham virus (JCV) who is undergoing immunosuppressive therapy. It addresses the risk of JCV activation and the associated development of progressive multifocal leukoencephalopathy (PML). The described approach aims to eliminate the JCV infection and eliminate the risk of JCV activation during immunosuppressive therapy.
The core gene editing strategy uses a CRISPR Cas9 endonuclease directed toward at least one target PAM sequence in the T-antigen of the JCV genome chosen from TTA, TTC, TTG, and TTT. The approach cleaves a target sequence in the JCV genome and disrupts the JCV genome, thereby eliminating the JCV infection. The patent also situates implementation within timing of immunosuppressive therapy before, during, or after administering the gene editing composition.
Additional disclosed subject matter includes pharmaceutical compositions comprising isolated nucleic acid encoding CRISPR components, expression vectors, and selected viral vector types. The disclosure also describes gRNA selection tied to JCV T-Ag encoding regions and SEQ ID NO sets, selected Cas9 variants and mutants, and broader CRISPR targetability considerations.
Claims Coverage
The claim set centers on CRISPR Cas9 cleavage of a T-antigen PAM-adjacent target sequence to disrupt the JCV genome, eliminate JCV infection, and eliminate the risk of JCV activation, with immunosuppressive therapy administered before, during, or after gene editing. Dependent claim refinements include latent infection screening, particular Cas9 variants, constrained gRNA selection tied to TM1/TM2/TM3 regions and specified SEQ ID NO sets, and specified viral vector types.
CRISPR Cas9 targeting PAM in JCV T-antigen
Administering to a subject latently infected with JCV an effective amount of a CRISPR Cas9 endonuclease directed toward at least one target PAM sequence in the T-antigen of the JCV genome chosen from TTA, TTC, TTG, and TTT.
Cleavage, disruption, and elimination of JCV infection
Cleaving the target sequence in the JCV genome, disrupting the JCV genome, eliminating the JCV infection, and eliminating the risk of JCV activation.
Immunosuppressive therapy timing relative to gene editing administration
Treating the subject with an immunosuppressive therapy at a time chosen from before, during, or after administering the gene editing composition.
Pre-treatment screening and verification of latent JCV infection
Screening a subject for latent JCV infection and verifying that a latent JCV infection is present prior to administering the treatment.
Selected Cas9 endonuclease variants and mutants
Selecting the CRISPR Cas9 endonuclease from specified Cas9 variants and mutants, including wild-type Cas9 and engineered or mutant Cas9 variants including nickase mutants and specific SpCas9/K mutants listed in the claim refinement.
gRNA selection from specified T-Ag regions and SEQ ID sets
Selecting at least one gRNA with a spacer complementary to a target sequence within TM1, TM2, or TM3 regions of the T-Ag encoding region of JCV DNA, where the chosen gRNAs come from SEQ ID NO: 1–4 (TM1), SEQ ID NO: 5–8 (TM2), SEQ ID NO: 9–12 (TM3), or any combination of these gRNAs.
Expression vectors of specified viral types
Selecting at least one expression vector from lentiviral, adenovirus, adeno-associated virus, VSV, pox virus, or retroviral vectors.
Overall, the inventive focus is CRISPR Cas9 targeting of the JCV T-antigen PAM sequence choices TTA, TTC, TTG, and TTT to cleave and disrupt the JCV genome, eliminate JCV infection, and eliminate the risk of JCV activation during immunosuppressive therapy, with dependent refinements for Cas9 variants, gRNA selection, latent infection verification, and expression vector types.
Stated Advantages
Eliminating the JCV infection.
Eliminating the risk of JCV activation in a subject undergoing immunosuppressive therapy.
Reducing the risk of JCV activation and the associated development of progressive multifocal leukoencephalopathy (PML).
Coordinating immunosuppressive therapy timing with gene editing administration.
Documented Applications
Treating a subject latently infected with JCV who is undergoing immunosuppressive therapy to eliminate the risk of JCV activation.
Reducing the risk of JCV activation in a subject latently infected with JCV undergoing immunosuppressive therapy.
A regimen context that includes natalizumab contingent on elimination of JCV infection and monitoring.
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