University of Massachusetts

Children's Medical Center Corporation

The General Hosptial Corporation

Mehmet Fatih Bolukbasi

Scot Andrew Wolfe

Erik J Sontheimer

Nadia Amrani

Ankit Gupta

Xandra O Breakefield

Daniel E Bauer

Arda Mizrak

Pengpeng Liu

Kevin Luk

Yuxuan Wu

Benjamin Roscoe

Jing Zeng

Okay Saydam

Provided herein are methods and compositions for increasing fetal hemoglobin levels in a cell by disrupting BCL11A expression at the genomic level. Also provided herein are methods and compositions relating to the treatment of hemoglobinopathies by reinduction of fetal hemoglobin levels.

Targeting BCL11A distal regulatory elements with a CAS9-CAS9 fusion for fetal hemoglobin reinduction

The present invention provides a Cas9 platform to facilitate single-site nuclease gene editing precision within a human genome. For example, a Cas9 nuclease/DNA-targeting unit (Cas9-DTU) fusion protein precisely delivers a Cas9/sgRNA complex to a specific target site within the genome for subsequent sgRNA-dependent cleavage of an adjacent target sequence. Alternatively, attenuating Cas9 binding using mutations to the a protospacer adjacent motif (PAM) recognition domain makes Cas9 target site recognition dependent on the associated DTU, all while retaining Cas9's sgRNA-mediated DNA cleavage fidelity. Cas9-DTU fusion proteins have improved target site binding precision, greater nuclease activity, and a broader sequence targeting range than standard Cas9 systems. Existing Cas9 or sgRNA variants (e.g., truncated sgRNAs (tru-gRNAs), nickases and FokI fusions) are compatible with these improvements to further reduce off-target cleavage. A robust, broadly applicable strategy is disclosed to impart Cas9 genome-editing systems with the single-genomic-site accuracy needed for safe, effective clinical application.

CAS9-CAS9 fusion proteins

Cas9-DNA targeting unit chimeras

Disclosed herein is an isolated nucleic acid molecule comprising a first nucleic acid sequence 5'-ACCCTGCCGCCTGGACTCCGCCTGT-3&#x2032; (SEQ ID NO: 22), or a functional variant thereof, operably linked to a second, heterologous nucleic acid sequence. The isolated nucleic acid molecule can be DNA (in an expression vector) and RNA (mRNA, shRNA, orncRNA). Also disclosed is a microvesicle comprising the nucleic acid molecule and a microvesicle preparation comprising the microvesicle. Also disclosed is an in vitro method of producing a microvesicle preparation enriched for a specific RNA sequence by transfecting cells with the nucleic acid sequence, and isolating microvesicles generated therefrom. Methods of delivering therapeutic RNA to a subject are also disclosed.

Targeting RNAs to microvesicles

Growing community of inventors

Worcester, MA, United States of America

Mehmet Fatih Bolukbasi