Compositions and methods for modulating gene expression
Inventors
Chen, Ci-Di • Zeldich, Ella • Abraham, Carmela
Assignees
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Publication Number
US-11807850-B2
Publication Date
2023-11-07
Expiration Date
Abstract
The present disclosure relates to compositions and methods for modulating gene expression and in particular to compositions and methods for increasing expression of Klotho. For example, the present disclosure provides methods for increasing expression of a Klotho gene in a human cell comprising introducing into the cell a CRISPR enzyme and a guide RNA comprising a guide sequence that is substantially complementary to a target sequence within or near the Klotho gene, wherein the guide RNA or the CRISPR enzyme associates with a transcriptional activation domain.
Core Innovation
The invention provides engineered CRISPR transcriptional activator constructs and related systems that include a CRISPR-dCas9 enzyme and a guide RNA. The guide RNA is configured to associate with an activation system comprising an adapter protein MS2 and a transcriptional activation domain comprising VP64, p65 and HSF1, to increase transcription and expression of the Klotho gene.
The guide RNA guide sequence is substantially complementary to a target sequence located within a region between the Klotho gene translation start site and 1 to 300 nucleotides upstream of the Klotho gene translation start site. The guide sequence is at least 95% identical to SEQ ID NO. 1 or SEQ ID NO. 3, and the guide RNA is associated with MS2 to recruit the activation domain in a targeted manner.
The disclosure also describes CRISPR transcriptional activation architectures in which dCas9-VP64 is used together with an MS2-p65-HSF1 scaffold arrangement, and an alternative scaffold arrangement using MS2 binding loops and an F6 aptamer to recruit MS2-VP64 fusions. The described systems provide multivalent transcriptional activation using adapter protein recruitment of activation domain fusions.
Claims Coverage
Independent claims cover four related inventive features centered on Klotho gene activation: a CRISPR-dCas9 system with a guide RNA targeted upstream of the Klotho translation start site, a guide RNA associated with MS2 and a VP64/p65/HSF1 transcriptional activation domain, a CRISPR-dCas9 transcriptional activation system, and the use of that activation system to increase expression in human neuronal or kidney cells or to treat a kidney disorder in a human subject.
Klotho gene upregulation in human neuronal or kidney cells
A method of increasing expression of a Klotho gene in a human neuronal or kidney cell by introducing a CRISPR-dCas9 enzyme and a guide RNA comprising a guide sequence at least 95% identical to SEQ ID NO. 1 or SEQ ID NO. 3, wherein the guide sequence is substantially complementary to a target sequence located within a region between the Klotho gene translation start site and 1 to 300 nucleotides upstream of the Klotho gene translation start site.
VP64/p65/HSF1 activation domain with MS2-associated guide RNA
The guide RNA associates with a transcriptional activation domain comprising VP64, p65 and HSF1, and the guide RNA is associated with the adapter protein MS2 to thereby increase expression of the Klotho gene.
CRISPR-dCas9 transcriptional activation system
A CRISPR-dCas9 enzyme is introduced, wherein the CRISPR enzyme comprises or is attached to a transcriptional activation domain comprising VP64, p65 and HSF1.
Kidney disorder treatment with MS2-associated Klotho activation
A method of treating a kidney disorder in a human subject by administering a CRISPR-dCas9 enzyme and a guide RNA comprising a guide sequence at least 95% identical to SEQ ID NO. 1 or SEQ ID NO. 3, wherein the guide sequence is substantially complementary to a target sequence located within a region between the Klotho gene translation start site and 1 to 300 nucleotides upstream of the Klotho gene translation start site.
The inventive coverage combines a CRISPR-dCas9 transcriptional activation system, a guide RNA with at least 95% identity to specified SEQ ID references that targets a region upstream of the Klotho translation start site, and MS2-associated recruitment of a VP64/p65/HSF1 activation domain to increase Klotho expression, with applications in human neuronal or kidney cells and in treating a kidney disorder in a human subject.
Stated Advantages
Increase expression of the Klotho gene.
Enable treatment of a kidney disorder in a human subject by increasing Klotho gene expression.
Treatment of a kidney disorder in a human subject by increasing expression of the Klotho gene.
Documented Applications
Increasing expression of a Klotho gene in a human neuronal or kidney cell.
Treating a kidney disorder in a human subject, including renal dysfunction, acute kidney injury, or kidney disease such as chronic kidney disease.
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