Allele-selective transcriptional repression of mutant HTT for the treatment of Huntington's disease.

Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin gene (HTT), which codes for the pathologic mutant HTT (mHTT) protein. Since normal HTT is thought to be important for brain function, we engineered zinc finger protein transcription factors (ZFP-TFs) to target the pathogenic CAG repeat and selectively lower mHTT as a therapeutic strategy. Using patient-derived fibroblasts and neurons, we demonstrate that ZFP-TFs selectively repress >99% of HD-causing alleles over a wide dose range while preserving expression of >86% of normal alleles. Other CAG-containing genes are minimally affected, and virally delivered ZFP-TFs are active and well tolerated in HD neurons beyond 100 days in culture and for at least nine months in the mouse brain. Using three HD mouse models, we demonstrate improvements in a range of molecular, histopathological, electrophysiological and functional endpoints. Our findings support the continued development of an allele-selective ZFP-TF for the treatment of HD.

Enhancing gene editing specificity by attenuating DNA cleavage kinetics.

Engineered nucleases have gained broad appeal for their ability to mediate highly efficient genome editing. However the specificity of these reagents remains a concern, especially for therapeutic applications, given the potential mutagenic consequences of off-target cleavage. Here we have developed an approach for improving the specificity of zinc finger nucleases (ZFNs) that engineers the FokI catalytic domain with the aim of slowing cleavage, which should selectively reduce activity at low-affinity off-target sites. For three ZFN pairs, we engineered single-residue substitutions in the FokI domain that preserved full on-target activity but showed a reduction in off-target indels of up to 3,000-fold. By combining this approach with substitutions that reduced the affinity of zinc fingers, we developed ZFNs specific for the TRAC locus that mediated 98% knockout in T cells with no detectable off-target activity at an assay background of ~0.01%. We anticipate that this approach, and the FokI variants we report, will enable routine generation of nucleases for gene editing with no detectable off-target activity.

Humanized Mouse Model of HIV-1 Latency with Enrichment of Latent Virus in PD-1+ and TIGIT+ CD4 T Cells.

Combination anti-retroviral drug therapy (ART) potently suppresses HIV-1 replication but does not result in virus eradication or a cure. A major contributing factor is the long-term persistence of a reservoir of latently infected cells. To study this reservoir, we established a humanized mouse model of HIV-1 infection and ART suppression based on an oral ART regimen. Similar to humans, HIV-1 levels in the blood of ART-treated animals were frequently suppressed below the limits of detection. However, the limited timeframe of the mouse model and the small volume of available samples makes it a challenging model with which to achieve full viral suppression and to investigate the latent reservoir. We therefore used an ex vivo latency reactivation assay that allows a semiquantitative measure of the latent reservoir that establishes in individual animals, regardless of whether they are treated with ART. Using this assay, we found that latently infected human CD4 T cells can be readily detected in mouse lymphoid tissues and that latent HIV-1 was enriched in populations expressing markers of T cell exhaustion, PD-1 and TIGIT. In addition, we were able to use the ex vivo latency reactivation assay to demonstrate that HIV-specific TALENs can reduce the fraction of reactivatable virus in the latently infected cell population that establishes in vivo, supporting the use of targeted nuclease-based approaches for an HIV-1 cure.IMPORTANCE HIV-1 can establish latent infections that are not cleared by current antiretroviral drugs or the body's immune responses and therefore represent a major barrier to curing HIV-infected individuals. However, the lack of expression of viral antigens on latently infected cells makes them difficult to identify or study. Here, we describe a humanized mouse model that can be used to detect latent but reactivatable HIV-1 in both untreated mice and those on ART and therefore provides a simple system with which to study the latent HIV-1 reservoir and the impact of interventions aimed at reducing it.

Diversifying the structure of zinc finger nucleases for high-precision genome editing.

Genome editing for therapeutic applications often requires cleavage within a narrow sequence window. Here, to enable such high-precision targeting with zinc-finger nucleases (ZFNs), we have developed an expanded set of architectures that collectively increase the configurational options available for design by a factor of 64. These new architectures feature the functional attachment of the FokI cleavage domain to the amino terminus of one or both zinc-finger proteins (ZFPs) in the ZFN dimer, as well as the option to skip bases between the target triplets of otherwise adjacent fingers in each zinc-finger array. Using our new architectures, we demonstrate targeting of an arbitrarily chosen 28 bp genomic locus at a density that approaches 1.0 (i.e., efficient ZFNs available for targeting almost every base step). We show that these new architectures may be used for targeting three loci of therapeutic significance with a high degree of precision, efficiency, and specificity.

Non-viral Delivery of Zinc Finger Nuclease mRNA Enables Highly Efficient In Vivo Genome Editing of Multiple Therapeutic Gene Targets.

It has previously been shown that engineered zinc finger nucleases (ZFNs) can be packaged into adeno-associated viruses (AAVs) and delivered intravenously into mice, non-human primates, and most recently, humans to induce highly efficient therapeutic genome editing in the liver. Lipid nanoparticles (LNPs) are synthetic delivery vehicles that enable repeat administration and are not limited by the presence of preexisting neutralizing antibodies in patients. Here, we show that mRNA encoding ZFNs formulated into LNP can enable >90% knockout of gene expression in mice by targeting the TTR or PCSK9 gene, at mRNA doses 10-fold lower than has ever been reported. Additionally, co-delivering mRNA-LNP containing ZFNs targeted to intron 1 of the ALB locus with AAV packaged with a promoterless human IDS or FIX therapeutic transgene can result in high levels of targeted integration and subsequent therapeutically relevant levels of protein expression in mice. Finally, we show repeat administration of ZFN mRNA-LNP after a single AAV donor dose results in significantly increased levels of genome editing and transgene expression compared to a single dose. These results demonstrate LNP-mediated ZFN mRNA delivery can drive highly efficient levels of in vivo genome editing and can potentially offer a new treatment modality for a variety of diseases.

Genome Editing in Neuroepithelial Stem Cells to Generate Human Neurons with High Adenosine-Releasing Capacity.

As a powerful regulator of cellular homeostasis and metabolism, adenosine is involved in diverse neurological processes including pain, cognition, and memory. Altered adenosine homeostasis has also been associated with several diseases such as depression, schizophrenia, or epilepsy. Based on its protective properties, adenosine has been considered as a potential therapeutic agent for various brain disorders. Since systemic application of adenosine is hampered by serious side effects such as vasodilatation and cardiac suppression, recent studies aim at improving local delivery by depots, pumps, or cell-based applications. Here, we report on the characterization of adenosine-releasing human embryonic stem cell-derived neuroepithelial stem cells (long-term self-renewing neuroepithelial stem [lt-NES] cells) generated by zinc finger nuclease (ZFN)-mediated knockout of the adenosine kinase (ADK) gene. ADK-deficient lt-NES cells and their differentiated neuronal and astroglial progeny exhibit substantially elevated release of adenosine compared to control cells. Importantly, extensive adenosine release could be triggered by excitation of differentiated neuronal cultures, suggesting a potential activity-dependent regulation of adenosine supply. Thus, ZFN-modified neural stem cells might serve as a useful vehicle for the activity-dependent local therapeutic delivery of adenosine into the central nervous system. Stem Cells Translational Medicine 2018;7:477-486.

Gene Correction of iPSCs from a Wiskott-Aldrich Syndrome Patient Normalizes the Lymphoid Developmental and Functional Defects.

Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency disease caused by mutations in the gene encoding the WAS protein (WASp). Here, induced pluripotent stem cells (iPSCs) were derived from a WAS patient (WAS-iPSC) and the endogenous chromosomal WAS locus was targeted with a wtWAS-2A-eGFP transgene using zinc finger nucleases (ZFNs) to generate corrected WAS-iPSC (cWAS-iPSC). WASp and GFP were first expressed in the earliest CD34(+)CD43(+)CD45(-) hematopoietic precursor cells and later in all hematopoietic lineages examined. Whereas differentiation to non-lymphoid lineages was readily obtained from WAS-iPSCs, in vitro T lymphopoiesis from WAS-iPSC was deficient with few CD4(+)CD8(+) double-positive and mature CD3(+) T cells obtained. T cell differentiation was restored for cWAS-iPSCs. Similarly, defects in natural killer cell differentiation and function were restored on targeted correction of the WAS locus. These results demonstrate that the defects exhibited by WAS-iPSC-derived lymphoid cells were fully corrected and suggests the potential therapeutic use of gene-corrected WAS-iPSCs.

Targeted gene addition in human CD34(+) hematopoietic cells for correction of X-linked chronic granulomatous disease.

Gene therapy with genetically modified human CD34(+) hematopoietic stem and progenitor cells (HSPCs) may be safer using targeted integration (TI) of transgenes into a genomic 'safe harbor' site rather than random viral integration. We demonstrate that temporally optimized delivery of zinc finger nuclease mRNA via electroporation and adeno-associated virus (AAV) 6 delivery of donor constructs in human HSPCs approaches clinically relevant levels of TI into the AAVS1 safe harbor locus. Up to 58% Venus(+) HSPCs with 6-16% human cell marking were observed following engraftment into mice. In HSPCs from patients with X-linked chronic granulomatous disease (X-CGD), caused by mutations in the gp91phox subunit of the NADPH oxidase, TI of a gp91phox transgene into AAVS1 resulted in ∼15% gp91phox expression and increased NADPH oxidase activity in ex vivo-derived neutrophils. In mice transplanted with corrected HSPCs, 4-11% of human cells in the bone marrow expressed gp91phox. This method for TI into AAVS1 may be broadly applicable to correction of other monogenic diseases.

Mammalian interspecies substitution of immune modulatory alleles by genome editing.

We describe a fundamentally novel feat of animal genetic engineering: the precise and efficient substitution of an agronomic haplotype into a domesticated species. Zinc finger nuclease in-embryo editing of the RELA locus generated live born domestic pigs with the warthog RELA orthologue, associated with resilience to African Swine Fever. The ability to efficiently achieve interspecies allele introgression in one generation opens unprecedented opportunities for agriculture and basic research.

Functional footprinting of regulatory DNA.

Regulatory regions harbor multiple transcription factor (TF) recognition sites; however, the contribution of individual sites to regulatory function remains challenging to define. We describe an approach that exploits the error-prone nature of genome editing-induced double-strand break repair to map functional elements within regulatory DNA at nucleotide resolution. We demonstrate the approach on a human erythroid enhancer, revealing single TF recognition sites that gate the majority of downstream regulatory function.

In vivo genome editing of the albumin locus as a platform for protein replacement therapy.

Site-specific genome editing provides a promising approach for achieving long-term, stable therapeutic gene expression. Genome editing has been successfully applied in a variety of preclinical models, generally focused on targeting the diseased locus itself; however, limited targeting efficiency or insufficient expression from the endogenous promoter may impede the translation of these approaches, particularly if the desired editing event does not confer a selective growth advantage. Here we report a general strategy for liver-directed protein replacement therapies that addresses these issues: zinc finger nuclease (ZFN) -mediated site-specific integration of therapeutic transgenes within the albumin gene. By using adeno-associated viral (AAV) vector delivery in vivo, we achieved long-term expression of human factors VIII and IX (hFVIII and hFIX) in mouse models of hemophilia A and B at therapeutic levels. By using the same targeting reagents in wild-type mice, lysosomal enzymes were expressed that are deficient in Fabry and Gaucher diseases and in Hurler and Hunter syndromes. The establishment of a universal nuclease-based platform for secreted protein production would represent a critical advance in the development of safe, permanent, and functional cures for diverse genetic and nongenetic diseases.

Correction of the sickle cell disease mutation in human hematopoietic stem/progenitor cells.

Sickle cell disease (SCD) is characterized by a single point mutation in the seventh codon of the ß-globin gene. Site-specific correction of the sickle mutation in hematopoietic stem cells would allow for permanent production of normal red blood cells. Using zinc-finger nucleases (ZFNs) designed to flank the sickle mutation, we demonstrate efficient targeted cleavage at the ß-globin locus with minimal off-target modification. By co-delivering a homologous donor template (either an integrase-defective lentiviral vector or a DNA oligonucleotide), high levels of gene modification were achieved in CD34(+) hematopoietic stem and progenitor cells. Modified cells maintained their ability to engraft NOD/SCID/IL2rγ(null) mice and to produce cells from multiple lineages, although with a reduction in the modification levels relative to the in vitro samples. Importantly, ZFN-driven gene correction in CD34(+) cells from the bone marrow of patients with SCD resulted in the production of wild-type hemoglobin tetramers.

Targeted correction and restored function of the CFTR gene in cystic fibrosis induced pluripotent stem cells.

Recently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources-potentially useful for the development of new therapeutic approaches. Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc-finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology-directed repair (HDR). We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other. The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells.

Improved specificity of TALE-based genome editing using an expanded RVD repertoire.

Transcription activator-like effector (TALE) proteins have gained broad appeal as a platform for targeted DNA recognition, largely owing to their simple rules for design. These rules relate the base specified by a single TALE repeat to the identity of two key residues (the repeat variable diresidue, or RVD) and enable design for new sequence targets via modular shuffling of these units. A key limitation of these rules is that their simplicity precludes options for improving designs that are insufficiently active or specific. Here we address this limitation by developing an expanded set of RVDs and applying them to improve the performance of previously described TALEs. As an extreme example, total conversion of a TALE nuclease to new RVDs substantially reduced off-target cleavage in cellular studies. By providing new RVDs and design strategies, these studies establish options for developing improved TALEs for broader application across medicine and biotechnology.

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

Parkinson's 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.

Editing T cell specificity towards leukemia by zinc finger nucleases and lentiviral gene transfer.

The transfer of high-avidity T cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted antigen specificities of the resultant TCRs. We designed zinc-finger nucleases (ZFNs) that promoted the disruption of endogenous TCR ß- and α-chain genes. Lymphocytes treated with ZFNs lacked surface expression of CD3-TCR and expanded with the addition of interleukin-7 (IL-7) and IL-15. After lentiviral transfer of a TCR specific for the Wilms tumor 1 (WT1) antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near purity and were superior at specific antigen recognition compared to donor-matched, unedited TCR-transferred cells. In contrast to unedited TCR-transferred cells, the TCR-edited lymphocytes did not mediate off-target reactivity while maintaining their anti-tumor activity in vivo, thus showing that complete editing of T cell specificity generates tumor-specific lymphocytes with improved biosafety profiles.

Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells.

Human induced pluripotent stem cells (iPSCs) represent a unique opportunity for regenerative medicine because they offer the prospect of generating unlimited quantities of cells for autologous transplantation, with potential application in treatments for a broad range of disorders. However, the use of human iPSCs in the context of genetically inherited human disease will require the correction of disease-causing mutations in a manner that is fully compatible with clinical applications. The methods currently available, such as homologous recombination, lack the necessary efficiency and also leave residual sequences in the targeted genome. Therefore, the development of new approaches to edit the mammalian genome is a prerequisite to delivering the clinical promise of human iPSCs. Here we show that a combination of zinc finger nucleases (ZFNs) and piggyBac technology in human iPSCs can achieve biallelic correction of a point mutation (Glu342Lys) in the α(1)-antitrypsin (A1AT, also known as SERPINA1) gene that is responsible for α(1)-antitrypsin deficiency. Genetic correction of human iPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene-targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences. Our results provide the first proof of principle, to our knowledge, for the potential of combining human iPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies.

In vivo genome editing restores haemostasis in a mouse model of haemophilia.

Editing of the human genome to correct disease-causing mutations is a promising approach for the treatment of genetic disorders. Genome editing improves on simple gene-replacement strategies by effecting in situ correction of a mutant gene, thus restoring normal gene function under the control of endogenous regulatory elements and reducing risks associated with random insertion into the genome. Gene-specific targeting has historically been limited to mouse embryonic stem cells. The development of zinc finger nucleases (ZFNs) has permitted efficient genome editing in transformed and primary cells that were previously thought to be intractable to such genetic manipulation. In vitro, ZFNs have been shown to promote efficient genome editing via homology-directed repair by inducing a site-specific double-strand break (DSB) at a target locus, but it is unclear whether ZFNs can induce DSBs and stimulate genome editing at a clinically meaningful level in vivo. Here we show that ZFNs are able to induce DSBs efficiently when delivered directly to mouse liver and that, when co-delivered with an appropriately designed gene-targeting vector, they can stimulate gene replacement through both homology-directed and homology-independent targeted gene insertion at the ZFN-specified locus. The level of gene targeting achieved was sufficient to correct the prolonged clotting times in a mouse model of haemophilia B, and remained persistent after induced liver regeneration. Thus, ZFN-driven gene correction can be achieved in vivo, raising the possibility of genome editing as a viable strategy for the treatment of genetic disease.

Efficient targeted gene disruption in the soma and germ line of the frog Xenopus tropicalis using engineered zinc-finger nucleases.

The frog Xenopus, an important research organism in cell and developmental biology, currently lacks tools for targeted mutagenesis. Here, we address this problem by genome editing with zinc-finger nucleases (ZFNs). ZFNs directed against an eGFP transgene in Xenopus tropicalis induced mutations consistent with nonhomologous end joining at the target site, resulting in mosaic loss of the fluorescence phenotype at high frequencies. ZFNs directed against the noggin gene produced tadpoles and adult animals carrying up to 47% disrupted alleles, and founder animals yielded progeny carrying insertions and deletions in the noggin gene with no indication of off-target effects. Furthermore, functional tests demonstrated an allelic series of activity between three germ-line mutant alleles. Because ZFNs can be designed against any locus, our data provide a generally applicable protocol for gene disruption in Xenopus.

Rapid and efficient clathrin-mediated endocytosis revealed in genome-edited mammalian cells.

Clathrin-mediated endocytosis (CME) is the best-studied pathway by which cells selectively internalize molecules from the plasma membrane and surrounding environment. Previous live-cell imaging studies using ectopically overexpressed fluorescent fusions of endocytic proteins indicated that mammalian CME is a highly dynamic but inefficient and heterogeneous process. In contrast, studies of endocytosis in budding yeast using fluorescent protein fusions expressed at physiological levels from native genomic loci have revealed a process that is very regular and efficient. To analyse endocytic dynamics in mammalian cells in which endogenous protein stoichiometry is preserved, we targeted zinc finger nucleases (ZFNs) to the clathrin light chain A and dynamin-2 genomic loci and generated cell lines expressing fluorescent protein fusions from each locus. The genome-edited cells exhibited enhanced endocytic function, dynamics and efficiency when compared with previously studied cells, indicating that CME is highly sensitive to the levels of its protein components. Our study establishes that ZFN-mediated genome editing is a robust tool for expressing protein fusions at endogenous levels to faithfully report subcellular localization and dynamics.