CRISPR therapy
Inventors
Duan, Dongsheng • HAKIM, CHADY • WASALA, NALINDA B. • Yue, Yongping
Assignees
University of Missouri St Louis • US Department of Health and Human Services
Publication Number
US-12152242-B2
Publication Date
2024-11-26
Expiration Date
2039-04-23
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Abstract
Disclosed herein are methods for systemically editing a gene in a subject and for systemically treating a genetic condition in a subject using a dual-vector CRISPR-Cas therapy. The methods comprise administering to the subject, via systemic administration, a gene editing AAV vector encoding a CRISPR effector protein (e.g., a Cas protein) and a targeting AAV vector providing one or more gRNAs targeted to the gene. In the methods, the ratio of the targeting AAV vector to the gene editing vector is greater than or equal to 2. Also provided are dual-vector systems for editing a gene or treating a genetic disease in a subject.
Core Innovation
The invention provides methods for systemically editing a gene in a subject using a dual-vector CRISPR-Cas system delivered by adeno-associated virus (AAV) vectors. The methods comprise administering to the subject via systemic delivery a gene editing AAV vector encoding a Cas gene under regulatory sequences directing its expression, and a targeting AAV vector providing one or more guide RNAs targeted to the gene. A critical feature is that the ratio of the targeting AAV vector to the gene editing AAV vector is greater than or equal to about 2:1. This systemic administration induces a double stranded break (DSB) in the target gene, which is repaired via non-homologous end joining (NHEJ), thereby achieving gene editing.
The problem addressed arises from the challenge of delivering a gene editing system systemically and continuously to restore therapeutic protein expression over an extended period throughout a subject. Previous methods delivered CRISPR components locally or transiently and did not demonstrate persistent, long-term systemic therapeutic effects. Notably, systemic administration of the dual-vector system faces the unexpected issue of selective degradation of the targeting vector providing the gRNA in vivo, resulting in failure of gene editing. This limits the potential for treating genetic disorders characterized by widespread loss of gene function. The invention solves this by employing a specific ratio with an excess of the targeting vector relative to the gene editing vector during systemic delivery.
The dual-vector system can be used to treat or ameliorate genetic disorders characterized by mutations that cause loss of protein expression or production of truncated nonfunctional proteins. The invention demonstrates utility in Duchenne muscular dystrophy (DMD), where systemic delivery of the CRISPR-Cas components restores dystrophin expression long-term in muscle tissue including cardiac muscle. The disclosed approach also contemplates targeting various genetic diseases by modifying the gRNAs to target other mutated genes, allowing for persistent, widespread gene correction through NHEJ-mediated gene editing without requiring donor DNA templates.
Claims Coverage
The patent discloses one independent claim defining a method for systemically editing a gene in a subject using a dual-vector CRISPR-Cas system and additional dependent claims specifying features related to genetic diseases, gene mutations, and vector details.
systemic gene editing with dual AAV vectors at a specific targeting to editing vector ratio
Administering systemically a mixture of a gene editing AAV vector encoding a Cas gene under regulatory control and a targeting AAV vector providing at least one gRNA targeted to the gene, wherein the ratio of targeting to gene editing vector is greater than or equal to 2:1, effecting gene editing via induction of a double stranded break repaired by non-homologous end joining (NHEJ).
treatment of genetic disorders caused by gene mutations
Systemically treating a genetic disorder caused by a gene mutation by delivering the dual AAV vectors targeting the mutated gene to restore gene function, including genetic diseases characterized by loss of protein expression or truncated nonfunctional proteins.
restoration of open reading frame and correction of frameshift mutations
Methods addressing frameshift mutations resulting in loss of open reading frame or premature stop codons, including deleting portions of the gene such as an exon containing the premature stop codon or inducing insertions/deletions to restore normal splicing signals.
use of dual-vector system without donor nucleic acid
The gene editing method excludes use of donor DNA sequences, relying solely on NHEJ-mediated repair without a repair template.
vector composition and promoters
Use of targeting AAV vectors comprising promoters driving gRNA expression and gene editing AAV vectors comprising ubiquitous or tissue-specific promoters and polyadenylation signals for Cas gene expression.
use of diverse Cas proteins
Gene editing AAV vectors encoding Cas proteins selected from major Cas types, preferably Cas9 or Cas12.
specified vector ratio ranges
The targeting to gene editing AAV vector ratio ranges from about 2:1 to about 10:1, preferably about 3:1.
applicability to multiple species
The methods apply to subjects including mice and humans.
The claims cover a method for systemic gene editing by co-administering dual AAV vectors encoding Cas and gRNA at a specified excess ratio of targeting vector to gene editing vector, enabling NHEJ-based gene correction without donor DNA for treating genetic disorders, applicable across Cas protein types and promoters, with preferred vector ratios and subject species.
Stated Advantages
The invention enables systemic and persistent gene editing leading to long-term therapeutic protein restoration.
The dual-vector approach addresses selective degradation of the targeting vector in systemic administration by optimizing vector ratios.
The strategy avoids the need for donor DNA templates, reducing risks of insertional mutagenesis and increasing efficiency in post-mitotic cells.
The methods provide a single treatment alternative that could lead to lifelong gene expression restoration.
Documented Applications
Systemic treatment of Duchenne muscular dystrophy (DMD) by restoring dystrophin expression via dual-vector CRISPR-Cas therapy.
Potential treatment of other inherited muscle diseases, lysosomal storage diseases, heritable connective tissue disorders, neurodegenerative disorders, and skeletal dysplasias by tailoring gRNAs to target mutated genes.
Systemic gene correction in mouse models, especially the mdx mouse model for DMD.
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