H. Steve Zhang

A TALE nuclease architecture for efficient genome editing

Nucleases that cleave unique genomic sequences in living cells can be used for targeted gene editing and mutagenesis. Here we develop a strategy for generating such reagents based on transcription activator–like effector (TALE) proteins from Xanthomonas. We identify TALE truncation variants that efficiently cleave DNA when linked to the catalytic domain of FokI and use these nucleases to generate discrete edits or small deletions within endogenous human NTF3 and CCR5 genes at efficiencies of up to 25%. We further show that designed TALEs can regulate endogenous mammalian genes. These studies demonstrate the effective application of designed TALE transcription factors and nucleases for the targeted regulation and modification of endogenous genes.

Generation of isogenic pluripotent stem cells differing exclusively at two early onset Parkinson point mutations

Patient-specific induced pluripotent stem cells (iPSCs) derived from somatic cells provide a unique tool for the study of human disease, as well as a promising source for cell replacement therapies. One crucial limitation has been the inability to perform experiments under genetically defined conditions. This is particularly relevant for late age onset disorders in which in vitro phenotypes are predicted to be subtle and susceptible to significant effects of genetic background variations. By combining zinc finger nuclease (ZFN)-mediated genome editing and iPSC technology, we provide a generally applicable solution to this problem, generating sets of isogenic disease and control human pluripotent stem cells that differ exclusively at either of two susceptibility variants for Parkinsons disease by modifying the underlying point mutations in the α-synuclein gene. The robust capability to genetically correct disease-causing point mutations in patient-derived hiPSCs represents significant progress for basic biomedical research and an advance toward hiPSC-based cell replacement therapies.

LRRK2 mutations cause mitochondrial DNA damage in iPSC-derived neural cells from Parkinson's disease patients: reversal by gene correction.

Parkinsons disease associated mutations in leucine rich repeat kinase 2 (LRRK2) impair mitochondrial function and increase the vulnerability of induced pluripotent stem cell (iPSC)-derived neural cells from patients to oxidative stress. Since mitochondrial DNA (mtDNA) damage can compromise mitochondrial function, we examined whether LRRK2 mutations can induce damage to the mitochondrial genome. We found greater levels of mtDNA damage in iPSC-derived neural cells from patients carrying homozygous or heterozygous LRRK2 G2019S mutations, or at-risk individuals carrying the heterozygous LRRK2 R1441C mutation, than in cells from unrelated healthy subjects who do not carry LRRK2 mutations. After zinc finger nuclease-mediated repair of the LRRK2 G2019S mutation in iPSCs, mtDNA damage was no longer detected in differentiated neuroprogenitor and neural cells. Our results unambiguously link LRRK2 mutations to mtDNA damage and validate a new cellular phenotype that can be used for examining pathogenic mechanisms and screening therapeutic strategies.

A designed zinc-finger transcriptional repressor of phospholamban improves function of the failing heart.

Selective inhibition of disease-related proteins underpins the majority of successful drug–target interactions. However, development of effective antagonists is often hampered by targets that are not druggable using conventional approaches. Here, we apply engineered zinc-finger protein transcription factors (ZFP TFs) to the endogenous phospholamban (PLN) gene, which encodes a well validated but recalcitrant drug target in heart failure. We show that potent repression of PLN expression can be achieved with specificity that approaches single-gene regulation. Moreover, ZFP-driven repression of PLN increases calcium reuptake kinetics and improves contractile function of cardiac muscle both in vitro and in an animal model of heart failure. These results support the development of the PLN repressor as therapy for heart failure, and provide evidence that delivery of engineered ZFP TFs to native organs can drive therapeutically relevant levels of gene repression in vivo. Given the adaptability of designed ZFPs for binding diverse DNA sequences and the ubiquity of potential targets (promoter proximal DNA), our findings suggest that engineered ZFP repressors represent a powerful tool for the therapeutic inhibition of disease-related genes, therefore, offering the potential for therapeutic intervention in heart failure and other poorly treated human diseases.Selective inhibition of disease-related proteins underpins the majority of successful drug-target interactions. However, development of effective antagonists is often hampered by targets that are not druggable using conventional approaches. Here, we apply engineered zinc-finger protein transcription factors (ZFP TFs) to the endogenous phospholamban (PLN) gene, which encodes a well validated but recalcitrant drug target in heart failure. We show that potent repression of PLN expression can be achieved with specificity that approaches single-gene regulation. Moreover, ZFP-driven repression of PLN increases calcium reuptake kinetics and improves contractile function of cardiac muscle both in vitro and in an animal model of heart failure. These results support the development of the PLN repressor as therapy for heart failure, and provide evidence that delivery of engineered ZFP TFs to native organs can drive therapeutically relevant levels of gene repression in vivo. Given the adaptability of designed ZFPs for binding diverse DNA sequences and the ubiquity of potential targets (promoter proximal DNA), our findings suggest that engineered ZFP repressors represent a powerful tool for the therapeutic inhibition of disease-related genes, therefore, offering the potential for therapeutic intervention in heart failure and other poorly treated human diseases.

A7 Drug discovery approach for rare neurological diseases: using novel zinc finger protein technology to develop potential therapy for huntington’s disease

There are approximately 7000 known rare and orphan diseases, over a third of which affect the central nervous system, virtually all do not have adequate treatment options. Shire is committed to developing innovative medicines to treat the fundamental biochemical abnormalities that result in pathologies caused by lysosomal storage disorders and other rare neurological diseases by selecting the right biological target based on extensive knowledge of disease pathophysiology and the right therapeutic modality from our array of technology platforms that includes antibodies, modified RNA, small molecules, gene therapy and protein therapeutics. This approach is particularly relevant for Huntington’s disease (HD), a rare and fatal neurodegenerative disease caused by a CAG trinucleotide repeat expansion in exon 1 of one copy of the Huntingtin (Htt) gene, resulting in expression of an aggregation-prone mutant protein. As this mutant protein is believed to be a primary cause of the pathophysiology in HD, Htt-lowering approaches are being explored using various technologies. Here, we will describe the use of an engineered zinc-finger protein transcription factor (ZFP TF) that preferentially down-regulates expression from the disease-causing copy of the Htt gene relative to the normal, unexpanded copy of the gene in both in vitro and in vivo HD models. Results presented here support the further development of allele-specific ZFP TFs as a potential therapy for HD.

43. Targeting the Biology of Heart Disease: Engineered Zinc Finger Protein Repressors of Phospholamban as a Potential Therapy for Congestive Heart Failure

Improper calcium handling of the heart is a hallmark of patients with congestive heart failure (CHF). Because calcium is critical for cardiac contractility, proteins that regulate calcium homeostasis are potential targets treating CHF. Phospholamban (PLN) decreases contractility by inhibiting the activity of Sarcoplasmic Reticulum Ca2+ ATPase 2 isoform A (SERCA2a); an increased PLN/SERCA2a ratio is often found in CHF patients. Recent studies have demonstrated that ablation or inhibition of PLN function can improve cardiac contractile properties in animal models of CHF, suggesting that down-regulation of PLN may improve cardiac function in CHF patients. Importantly, inhibition of PLN enhances calcium handling without activating b-adrenergic pathways, which is known to have many side-effects and increase mortality. The development of small-molecule inhibitors of PLN function has so far been unsuccessful, largely due to the difficulty of inhibiting protein-protein interactions (such as that between PLN and SERCA2a) using small molecules. On the other hand, approaches that aim to block the expression of PLN may provide a superior means of achieving the desired therapeutic effect.