S. Kaye Spratt

Induction of angiogenesis in a mouse model using engineered transcription factors

The relationship between the structure of zinc-finger protein (ZFP) transcription factors and DNA sequence binding specificity has been extensively studied. Advances in this field have made it possible to design ZFPs de novo that will bind to specific targeted DNA sequences. It has been proposed that such designed ZFPs may eventually be useful in gene therapy. A principal advantage of this approach is that activation of an endogenous gene ensures expression of the natural array of splice variants. Preliminary studies in tissue culture have validated the feasibility of this approach. The studies reported here were intended to test whether engineered transcription factors are effective in a whole-organism model. ZFPs were designed to regulate the endogenous gene encoding vascular endothelial growth factor-A (Vegfa). Expression of these new ZFPs in vivo led to induced expression of the protein VEGF-A, stimulation of angiogenesis and acceleration of experimental wound healing. In addition, the neovasculature resulting from ZFP-induced expression of Vegfa was not hyperpermeable as was that produced by expression of murine Vegfa164 cDNA. These data establish, for the first time, that specifically designed transcription factors can regulate an endogenous gene in vivo and evoke a potentially therapeutic biophysiologic effect.

Synthetic Zinc Finger Transcription Factor Action at an Endogenous Chromosomal Site ACTIVATION OF THE HUMAN ERYTHROPOIETIN GENE

We have targeted the activation of an endogenous chromosomal locus including the human erythropoietin gene using synthetic transcription factors. These transcription factors are targeted to particular DNA sequences in the 5′-flanking region of the erythropoietin gene through engineering of a zinc finger DNA binding domain. The DNA binding domain is linked to a VP16 transcriptional activation domain. We find that these synthetic transcription factors invariably activate transiently transfected templates in which sequences within the 5′ flank of the erythropoietin gene are fused to a luciferase reporter. The efficiency of activation under these circumstances at a defined site is dependent on DNA binding affinity. In contrast, only a subset of these same zinc finger proteins is able to activate the endogenous chromosomal locus. The activity of these proteins is influenced by their capacity to gain access to their recognition elements within the chromatin infrastructure. Zinc finger transcription factors will provide a powerful tool to probe the determinants of chromatin accessibility and remodeling within endogenous chromosomal loci.

Zinc-finger protein-targeted gene regulation: Genomewide single-gene specificity

Zinc-finger protein transcription factors (ZFP TFs) can be designed to control the expression of any desired target gene, and thus provide potential therapeutic tools for the study and treatment of disease. Here we report that a ZFP TF can repress target gene expression with single-gene specificity within the human genome. A ZFP TF repressor that binds an 18-bp recognition sequence within the promoter of the endogenous CHK2 gene gives a >10-fold reduction in CHK2 mRNA and protein. This level of repression was sufficient to generate a functional phenotype, as demonstrated by the loss of DNA damage-induced CHK2-dependent p53 phosphorylation. We determined the specificity of repression by using DNA microarrays and found that the ZFP TF repressed a single gene (CHK2) within the monitored genome in two different cell types. These data demonstrate the utility of ZFP TFs as precise tools for target validation, and highlight their potential as clinical therapeutics.

Efficient clinical scale gene modification via zinc finger nuclease-targeted disruption of the HIV co-receptor CCR5

Since HIV requires CD4 and a co-receptor, most commonly C-C chemokine receptor 5 (CCR5), for cellular entry, targeting CCR5 expression is an attractive approach for therapy of HIV infection. Treatment of CD4(+) T cells with zinc-finger protein nucleases (ZFNs) specifically disrupting chemokine receptor CCR5 coding sequences induces resistance to HIV infection in vitro and in vivo. A chimeric Ad5/F35 adenoviral vector encoding CCR5-ZFNs permitted efficient delivery and transient expression following anti-CD3/anti-CD28 costimulation of T lymphocytes. We present data showing CD3/CD28 costimulation substantially improved transduction efficiency over reported methods for Ad5/F35 transduction of T lymphocytes. Modifications to the laboratory scale process, incorporating clinically compatible reagents and methods, resulted in a robust ex vivo manufacturing process capable of generating >10(10) CCR5 gene-edited CD4+ T cells from healthy and HIV+ donors. CD4+ T-cell phenotype, cytokine production, and repertoire were comparable between ZFN-modified and control cells. Following consultation with regulatory authorities, we conducted in vivo toxicity studies that showed no detectable ZFN-specific toxicity or T-cell transformation. Based on these findings, we initiated a clinical trial testing the safety and feasibility of CCR5 gene-edited CD4+ T-cell transfer in study subjects with HIV-1 infection.

An engineered transcription factor which activates VEGF-A enhances recovery after spinal cord injury.

Spinal cord injury (SCI) leads to local vascular disruption and progressive ischemia, which contribute to secondary degeneration. Enhancing angiogenesis through the induction of vascular endothelial growth factor (VEGF)-A expression therefore constitutes an attractive therapeutic approach. Moreover, emerging evidence suggests that VEGF-A may also exhibit neurotrophic, neuroprotective, and neuroproliferative effects. Building on this previous work, we seek to examine the potential therapeutic benefits of an engineered zinc finger protein (ZFP) transcription factor designed to activate expression of all isoforms of endogenous VEGF-A (ZFP-VEGF). Administration of ZFP-VEGF resulted in increased VEGF-A mRNA and protein levels, an attenuation of axonal degradation, a significant increase in vascularity and decreased levels of apoptosis. Furthermore, ZFP-VEGF treated animals showed significant improvements in tissue preservation and neurobehavioural outcomes. These data suggest that activation of VEGF-A via the administration of an engineered ZFP transcription factor holds promise as a therapy for SCI and potentially other forms of neurotrauma.

Engineered Zinc Finger–Activating Vascular Endothelial Growth Factor Transcription Factor Plasmid DNA Induces Therapeutic Angiogenesis in Rabbits With Hindlimb Ischemia

Background—Therapeutic angiogenesis seeks to promote blood vessel growth to improve tissue perfusion. Vascular endothelial growth factor (VEGF) exists in multiple isoforms. We investigated an engineered zinc finger–containing transcription factor plasmid designed to activate the endogenous VEGF gene (ZFP-VEGF). Methods and Results—New Zealand White rabbits (n=56) underwent unilateral femoral artery ligation and excision. At day 10 postoperatively, the ischemic muscle received ZFP treatment (500 &mgr;g ZFP-VEGF plasmid) or no ZFP treatment (&bgr;-galactosidase, empty, or no plasmid). Group 1 (n=13) was harvested 3 days after injection to examine VEGF mRNA by real-time polymerase chain reaction and protein by ELISA. Groups 2 (n=13) and 3 (n=10) were harvested 11 days after injection. Group 2 was studied by histology and group 3, by histology and changes in blood flow. Groups 4 and 5 (n=10 each) were harvested 22 and 32 days after injection, respectively, and studied for changes in blood flow. In group 1, VEGF mRNA copy numbers were significantly higher for VEGF121, VEGF165, VEGF189, and protein in the ZFP-VEGF-treatment versus no-ZFP-treatment arms. In groups 2 and 3, capillary density and proliferating cells were significantly greater and apoptosis significantly lower in the treatment versus no-treatment arms. Changes in the blood flow ratio of the ischemic to the nonischemic limb were significantly greater in the treatment versus no-ZFP-treatment groups (6.57±1.52% versus 3.38±0.87%, P<0.005; 13.15±1.77% versus 6.13±1.55%, P<0.001; and 20.16±2.84% versus 13.88±3.14%, P<0.01, for groups 3, 4, and 5, respectively). Conclusions—This engineered ZFP-VEGF–activating transcription factor may provide a novel approach to treat peripheral arterial disease.

Gene Transfer of an Engineered Transcription Factor Promoting Expression of VEGF-A Protects Against Experimental Diabetic Neuropathy

Peripheral neuropathy is a common, irreversible complication of diabetes. We investigated whether gene transfer of an engineered zinc finger protein transcription factor (ZFP-TF) designed to upregulate expression of the endogenous vascular endothelial growth factor (VEGF)-A gene could protect against experimental diabetic neuropathy. ZFP-TF–driven activation of the endogenous gene results in expression of all of the VEGF-A isoforms, a fact that may be of significance for recapitulation of the proper biological responses stimulated by this potent neuroprotective growth factor. We show here that this engineered ZFP-TF activates VEGF-A in appropriate cells in culture and that the secreted VEGF-A protein induced by the ZFP protects neuroblastoma cell lines from a serum starvation insult in vitro. Importantly, single and repeat intramuscular injections of formulated plasmid DNA encoding the VEGF-A–activating ZFP-TF resulted in protection of both sensory and motor nerve conduction velocities in a streptozotocin-induced rat model of diabetes. These data suggest that VEGF-A–activating ZFP-TFs may ultimately be of clinical utility in the treatment of this disease.

Selective uptake and sustained expression of AAV vectors following subcutaneous delivery

Recombinant adeno‐associated viral (rAAV) vectors are capable of long‐term expression of secreted and intracellular proteins following delivery to muscle, liver, and the central nervous system. In this study, we have evaluated subcutaneous injection of rAAV encoding a variety of transgenes as an alternative route of administration for the systemic delivery of therapeutic proteins.

Treatment of Traumatic Brain Injury Using Zinc-Finger Protein Gene Therapy Targeting VEGF-A

Vascular endothelial growth factor (VEGF) plays a role in angiogenesis and has been shown to be neuroprotective following central nervous system trauma. In the present study we evaluated the pro-angiogenic and neuroprotective effects of an engineered zinc-finger protein transcription factor transactivator targeting the vascular endothelial growth factor A (VEGF-ZFP). We used two virus delivery systems, adeno-virus and adeno-associated virus, to examine the effects of early and delayed VEGF-A upregulation after brain trauma, respectively. Male Sprague-Dawley rats were subject to a unilateral fluid percussion injury (FPI) of moderate severity (2.2-2.5 atm) followed by intracerebral microinjection of either adenovirus vector (Adv) or an adeno-associated vector (AAV) carrying the VEGF-ZFP construct. Adv-VEGF-ZFP-treated animals had significantly fewer TUNEL positive cells in the injured penumbra of the cortex (p<0.001) and hippocampus (p=0.001) relative to untreated rats at 72 h post-injury. Adv-VEGF-ZFP treatment significantly improved fEPSP values (p=0.007) in the CA1 region relative to injury alone. Treatment with AAV2-VEGF-ZFP resulted in improved post-injury microvascular diameter and improved functional recovery on the balance beam and rotarod task at 30 days post-injury. Collectively, the results provide supportive evidence for the concept of acute and delayed treatment following TBI using VEGF-ZFP to induce angiogenesis, reduce cell death, and enhance functional recovery.

Intramuscular administration of a VEGF zinc finger transcription factor activator (VEGF-ZFP-TF) improves functional outcomes in SOD1 rats.

Abstract Amyotrophic lateral sclerosis (ALS) is characterized by motor neuron loss leading to paralysis and death. Vascular endothelial growth factor (VEGF) has angiogenic, neurotrophic, and neuroprotective properties, and has preserved neuromuscular function and protected motor neurons in rats engineered to overexpress the human gene coding the mutated G93A form of the superoxide dismutase-1 (SOD1). We assessed the effects of intramuscular administration of a plasmid that encodes a zinc finger protein transcription factor (ZFP-TF) engineered to induce VEGF expression in the SOD1 rat model of ALS. Weekly injections of the plasmid preserved ipsilateral hindlimb grip strength and markedly improved rotarod performance in SOD1 rats compared to the vehicle-treated group. The number of motor neurons and the proportion of innervated neuromuscular junctions were similar in both groups. In conclusion, our data suggest that administration of the VEGF-ZFP-TF may be neuroprotective and has potential as a safe and practical approach for the management of motor disability in ALS.