Developing Epigenetic Therapies for Haploinsufficiency

The rapid development of genome editing technologies in the last couple of decades has opened the door for the creation of novel therapeutics and cures for genetic disorders that were once thought to be untreatable. In this dissertation, we explore the potential to develop these tools for the treatment of three genetic disorders, Neurofibromatosis type 1 (NF1), Syngap1, and 22q11.2 deletion syndrome (22q11.2 DS). Although these disorders are very different from one another in terms of gene targets and patient presentation, they share a common theme: they are all a type of haploinsufficiency where an epigenetic gene therapy may provide immense benefit to patients.Epigenetic editing using CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) involves modifying the chemical modifications that control gene expression without altering the underlying DNA sequence. CRISPR is a powerful tool for editing the genome, which involves using Cas proteins and their ability to complex with RNA guides to localize to specific regions of the genome. The CRISPR system has been adapted to target epigenetic marks using modified versions of the Cas proteins and fusing it to an epigenetic enzyme that adds or removes chemical marks on DNA or histones, such as DNA methyltransferase or histone deacetylase, respectively. This modified Cas9 protein can be directed to specific regions of the genome using guide RNAs that are designed to target specific sequences. Alternatively, CRISPR can also be used to recruit proteins that activate or repress gene expression to specific regions of the genome. This involves fusing a modified Cas9 protein to a transcriptional activator or repressor protein respectively, which can then be targeted to specific regions of the genome using guide RNAs. Overall, epigenetic editing using CRISPR provides a powerful tool for understanding the role of epigenetic modifications in gene regulation and has potential therapeutic applications in treating diseases that involve aberrant epigenetic marks. In summary, for NF1, we utilized a duplex CRISPR repression system to downregulate disease specific genes, and for Syngap1 and 22q11.2 deletion syndrome, we utilized a CRISPR activation system to upregulate disease specific genes

Quantifying Genome-Editing Outcomes at Endogenous Loci with SMRT Sequencing

Targeted genome editing with engineered nucleases has transformed the ability to introduce precise sequence modifications at almost any site within the genome. A major obstacle to probing the efficiency and consequences of genome editing is that no existing method enables the frequency of different editing events to be simultaneously measured across a cell population at any endogenous genomic locus. We have developed a method for quantifying individual genome-editing outcomes at any site of interest with single-molecule real-time (SMRT) DNA sequencing. We show that this approach can be applied at various loci using multiple engineered nuclease platforms, including transcription-activator-like effector nucleases (TALENs), RNA-guided endonucleases (CRISPR/Cas9), and zinc finger nucleases (ZFNs), and in different cell lines to identify conditions and strategies in which the desired engineering outcome has occurred. This approach offers a technique for studying double-strand break repair, facilitates the evaluation of gene-editing technologies, and permits sensitive quantification of editing outcomes in almost every experimental system used

Author Correction: Gene correction for SCID-X1 in long-term hematopoietic stem cells.

The original version of this Article omitted the following from the Acknowledgements: G.B. acknowledges the support from the Cancer Prevention and Research Institute of Texas (RR140081 and RR170721).This has now been corrected in both the PDF and HTML versions of the Article

Gene correction for SCID-X1 in long-term hematopoietic stem cells.

Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. We achieve high levels of targeting frequencies (median 45%) in CD34+ HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect in a patient derived HSPC population in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl

Author Correction: Gene correction for SCID-X1 in long-term hematopoietic stem cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Gene correction for SCID-X1 in long-term hematopoietic stem cells

Gene correction in hematopoietic stem cells could be a powerful way to treat monogenic diseases of the blood and immune system. Here the authors develop a strategy using CRISPR-Cas9 and an aAdeno-Associated vVirus(AAV)-delivered IL2RG cDNA to correct X-linked sSevere Ccombined iImmunodeficiency (SCID-X1) with a high success rate

Author Correction: Gene correction for SCID-X1 in long-term hematopoietic stem cells

The original version of this Article omitted the following from the Acknowledgements: “G.B. acknowledges the support from the Cancer Prevention and Research Institute of Texas (RR140081 and RR170721).”This has now been corrected in both the PDF and HTML versions of the Article

Gene correction for SCID-X1 in long-term hematopoietic stem cells.

Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. We achieve high levels of targeting frequencies (median 45%) in CD34+ HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect in a patient derived HSPC population in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl