Gene Therapy Approaches For Treatment Research Articles

A Novel Recombinant Eklf-GATA1 Fusion Protein Reduces Sickling of Erythrocytes Cultured from CD34+ Cells with Sickle Cell Disease

Current gene therapy approaches for treatment of hemoglobinopathies involve viral transduction of hematopoietic stem cells with antisickling globin genes. Hemoglobin A2 (HA2, α2δ2), expressed at a low level due to the lack of Eklf binding motif in its promoter region, is fully functional and could be a valid anti-sickling agent in sickle cell disease, as well as a substitute of hemoglobin A in β-thalassemia. We had previously demonstrated that two Eklf-GATA1 fusion proteins could significantly activate δ-globin expression in CD34+ cells from healthy and sickle trait donor’s blood. Here we report the effects of Eklf-GATA1 on hemoglobin expression and phenotypic correction using erythrocytes cultured from CD34+ cells with sickle cell disease. We found that enforced expression of Eklf-GATA1 fusion protein enhanced globin gene expression in the erythrocytesas compared with vector control. The long-form Eklf-GATA1 up-regulated β-globin gene expression 2.0-fold, δ-globin gene expression 4.3-fold, and γ-globin gene expression 2.6-fold. The medium-form EKLF-GATA1 up-regulated δ-globin gene expression 2.3-fold and γ-globin 1.3-fold, but had no significant effect on β-globin gene expression. HPLC revealed a percentage of HA2+HbF was increased from 8.1 % in vector-transduced cells to 19.7% in medium-form Eklf-GATA-transduced-cells (p<0.01) and 14.4% in long-form Eklf-GATA-transduced-cells (p<0.01). Upon deoxygenation, the percentage of sickling erythrocyte was lower to 79.8% in medium-form Eklf-GATA-transduced cells as compared with 89.8% in vector-transduced-cells (p<0.05). Flow cytometry analyses of CD71/GPA and thiazole orange staining indicated that erythroid cell differentiation and enucleation were not affected by Eklf-GATA1. Our results shown that long form Eklf-GATA1 fusion protein has major effects on d- and g-globin induction than β-globin; the medium form Eklf-GATA1 elevated δ- and γ-globin expression without an effect on β-globin expression. Our results indicate that these fusion constructs could be a valuable genetic therapeutic tool for hemoglobinopathies, and warrant further preclinical study and evaluation. DisclosuresNo relevant conflicts of interest to declare.

Binding of DNA with Abf2p Increases Efficiency of DNA Uptake by Isolated Mitochondria.

Mutations in mitochondrial DNA often lead to severe hereditary diseases that are virtually resistant to symptomatic treatment. During the recent decades, many efforts were made to develop gene therapy approaches for treatment of such diseases using nucleic acid delivery into the organelles. The possibility of DNA import into mitochondria has been shown, but this process has low efficiency. In the present work, we demonstrate that the efficiency of DNA import can be significantly increased by preforming its complex with a mitochondria-targeted protein nonspecifically binding with DNA. As a model protein, we used the yeast protein Abf2p. In addition, we measured the length of the DNA site for binding this protein and the dissociation constant of the corresponding DNA-protein complex. Our data can serve as a basis for development of novel, highly efficient approaches for suppressing mutations in the mitochondrial genome.

186. Translating an Optogenetic Gene Therapy Approach for Treatment of Neuropathic Pain in Humans

Optogenetics has been established as a powerful tool to study the central and peripheral nervous systems with its potential for directly treating human disease tantalizingly on the horizon. Optogenetic inhibition of pain in mice has been shown to be effective and provides an attractive initial application. We aim to translate this approach to treat neuropathic pain in humans. Firstly, we demonstrated that the inhibitory opsin, NpHR, delivered by intraneural injections of AAV6 could reduce pain (mechanical allodynia) in a clinically meaningful paradigm i.e. vector delivery after establishment of neuropathic pain in the chronic constriction injury (CCI) model. The inhibitory opsin, iC1C2 (a chloride channel requiring less light than NpHR to pass currents) could also reduce pain in this system. We next tested a surgical approach more amenable to patients than nerve injection. Lumbar puncture is a routine clinical practice and AAV8 delivery by this method results in efficient gene delivery to sensory neurons in rodents, dogs and pigs. We found that intrathecal administration of AAV8 expressing iC1C2 could decrease mechanical allodynia in the CCI mouse model. The magnitude of pain inhibition correlated with transduction levels and only 10% transduction of sensory neurons was required for pain relief. To further validate this approach we utilized a second rodent pain model with translational relevance. The mouse tibia fracture/cast immobilization model of Complex Regional Pain Syndrome (CRPS) presents extreme allodynia and recapitulates human disease effectively. We found that intrathecal delivery of AAV8 encoding iC1C2 following development of the CRPS model resulted in light-mediated pain inhibition. Taken together, our results confirm optogenetic therapy for neuropathic pain as an attractive clinical application of this technology.

551. Enhancement of Gene Therapy Treatment for Sandhoff Disease Through Complimentary Drug Therapy

GM2 gangliosidoses is a group of neurodegenerative lysosomal storage disorders caused by deficiency in the β-hexosaminidase A (HexA) enzyme. HexA is a heterodimer composed of 2 subunits; α- (encoded by the HEXA gene) and β-hexosaminidase β (Encoded by the HEXB gene). Mutations in either gene may cause inactivity of HexA leading to either Tay-Sachs disease (TSD, HEXA mutation) or Sandhoff disease (SD, HEXB mutation) respectively. TSD and SD are clinically indistinguishable phenotypes principally affecting infants and young children that are fatal before the age of 4 years; there is currently no effective available treatment. A mouse model for SD has been developed and these mice reach a humane end point at 14-17 weeks of age. Gene therapy can be an important primary therapeutic strategy, but may fall short of a complete rescue. Neuroinflammation and neurodegeneration have been identified as hallmark pathological mechanisms in GM2 gangliosidosis and can be adjunctive therapeutic targets. Neuroanti-inflammatory/neuroprotective agents like non-steroidal anti-inflammatory drugs (NSAIDs), Histone deacetylase inhibitors and pharmacological chaperones have shown ameliorating effects in SD and similar disease models when used alone. Adeno associated virus (AAV) based expression of Hexosaminidase isoenzymes has been shown to increase survival of Sandhoff mice for long term. We tested the combined role of gene therapy and neuroanti-inflammatory/neuroprotective agents in SD mice. Our methods include injecting neonatal SD mice with a relatively low dose (2×1013 vg/kg) of a novel AAV9 vector expressing Hex A using both α and β subunits. A pilot study established the survival of these mice to be approximately 24 weeks, a 55% increase in life span over vehicle-injected controls. We observed a 47% increase in Hex A activity in the midbrain of treated mice as compared to the vehicle injected controls. Cohorts received treatment with neonatal gene therapy alone or in combination with indomethacin (a NSAID), pyrimethamine (a proven pharmacological chaperone for Hex A) and ITF2357 (a histone deactylase inhibitor) to elicit their combinational therapeutic potential. Each drug is administered daily via oral gavage starting the age of 6 weeks. All treatments have been completed and mice are being monitored for survival benefit and locomotor behaviour. Further analyses of enzyme activity, GM2 ganglioside levels and copy numbers will be done. The results of this study will establish a proof-of-concept for a novel combination gene therapy approach for treatment of GM2 gangliosidoses and similar disorders.

A Novel Recombinant Eklf-GATA1 Fusion Protein Reduces Erythrocytes Sickling in Human Erythroid Culture Model

Current gene therapy approaches for treatment of hemoglobinopathies involve viral transduction of hematopoietic stem cells with "antisickling" globin genes. Hemoglobin A2 (HbA2, α2δ2), expressed at a low level due to the lack of Eklf binding motif in its promoter region, is fully functional and could be a valid substitute for hemoglobin A in β-thalassemia, as well as an "anti-sickling" agent in sickle cell disease. We have previously demonstrated that two Eklf-GATA1 fusion proteins, which were recruited to the GATA1 binding motif at the δ-globin promoter, can significantly activate δ-globin expression in K562 cells and hematopoietic stem CD34+ cells. Here, we report that enforced expression of Eklf-GATA1 fusion protein in sickle trait CD34+ cells significantly increased δ-globin expression, as determined by quantitative PCR and HPLC. Upon deoxygenation, the percentage of sickling cells was lower in Eklf-GATA1-transduced red blood cells as compared with mock-transduced cells. By a series of flow cytometry analyses, which includes BRDU incorporation assay, CD71/GPA, and thiazole orange staining, we found that erythroid cells proliferation, differentiation and enucleation were not affected by Eklf-GATA1 expression. To assess the potential off-target effects of our fusion protein constructs, we analyzed differentially expressed genes (and proteins) in vector-only and Eklf-GATA-1 transduced-CD34+ cells by microarray and proteomic changes by liquid chromatography-mass spectrometry (LC-MS). We found that over-expression of Eklf-GATA1 resulted in a less than 2-fold change in the gene expression profile related to bone marrow hematopoiesis, and there were no significant changes that were detected in proteomic profiling. These results indicate that these fusion constructs could be a valuable genetic therapeutic tool for hemoglobinopathies, and warrant further preclinical study and evaluation. DisclosuresNo relevant conflicts of interest to declare.

Oligo/Polynucleotide-Based Gene Modification: Strategies and Therapeutic Potential

Oligonucleotide- and polynucleotide-based gene modification strategies were developed as an alternative to transgene-based and classical gene targeting-based gene therapy approaches for treatment of genetic disorders. Unlike the transgene-based strategies, oligo/polynucleotide gene targeting approaches maintain gene integrity and the relationship between the protein coding and gene-specific regulatory sequences. Oligo/polynucleotide-based gene modification also has several advantages over classical vector-based homologous recombination approaches. These include essentially complete homology to the target sequence and the potential to rapidly engineer patient-specific oligo/polynucleotide gene modification reagents. Several oligo/polynucleotide-based approaches have been shown to successfully mediate sequence-specific modification of genomic DNA in mammalian cells. The strategies involve the use of polynucleotide small DNA fragments, triplex-forming oligonucleotides, and single-stranded oligodeoxynucleotides to mediate homologous exchange. The primary focus of this review will be on the mechanistic aspects of the small fragment homologous replacement, triplex-forming oligonucleotide-mediated, and single-stranded oligodeoxynucleotide-mediated gene modification strategies as it relates to their therapeutic potential.

Regulatory Considerations for Translating Gene Therapy: A European Union Perspective

A preclinical study on a gene therapy approach for treatment of the severe muscle weakness associated with a variety of neuromuscular disorders provides a forum to discuss the translational challenges of gene therapy from a regulatory point of view. In this Perspective, the findings are considered from the view of European regulatory requirements for first clinical use.

266. Development of rAAV-Based Skeletal and Cardiac Muscle Regulatory Cassettes for Gene Therapy of Duchenne Muscular Dystrophy

Top of pageAbstract A promising gene therapy approach for treatment of DMD involves systemic delivery of rAAV vectors encoding microdystrophin. This method was shown to mediate efficient transduction of all striated muscles and high-level transgene expression driven by the ubiquitous CMV promoter. However, the use of viral promoters increases the risk of an immune response, largely due to transgene expression in antigen-presenting cells, and may thus be inadequate for long-term therapy. An ideal regulatory cassette for gene therapy of DMD thus needs to direct high-level, tissue-specific expression in skeletal and cardiac muscle and be short enough to package into the rAAV microdystrophin construct. Our lab has developed a series of muscle-specific regulatory cassettes based on the murine muscle creatine kinase (MCK) gene enhancer and promoter regions. The previous |[ldquo]|best|[rdquo]| small cassette, CK6 (580 bp) drives microdystrophin expression in skeletal muscle following systemic rAAV delivery, at 10% of the level of the CMV promoter. However, expression in heart and diaphragm is very low. To improve expression, we combined the wild type MCK enhancer with a 190 bp enhancer region from the murine alpha myosin heavy chain (MyHC) gene, which is expressed at high levels in cardiac muscle. Addition of the MyHC enhancer increased activity in MM14 skeletal myocytes by 5-fold as assessed by alkaline phosphatase reporter gene activity. Furthermore, high-level expression was detected in skeletal muscle, as well as heart and diaphragm after systemic delivery in mice of rAAV6 vectors encoding these constructs. The activity of the hybrid cassette was 7-fold higher in heart and 3-fold higher in diaphragm and soleus (predominantly slow-twitch muscle fibers) when compared to the wild type MCK cassette, while the activity in tibialis anterioris (predominantly fast-twitch muscle fibers) was unchanged. Expression was restricted to muscle tissue, as evidenced by nearly undetectable levels in the liver, spleen, lung, and aorta. Additional cassettes comprising the MyHC enhancer and various modifications of the minimal MCK cassette have also been tested in MM14 skeletal myocytes. The strongest cassettes were shown to possess 12-fold higher activity than the wild type MCK cassette, and were about 55% as active as the CMV promoter. These contained a 63 bp deletion within the MCK enhancer, addition of a 50 bp region downstream of the transcription initiation site, and a mutation that creates a consensus initiator-binding site from the terminal transferase promoter. We are currently testing activity of these constructs in mouse muscle and non-muscle tissues following systemic delivery of rAAV6 vectors. The strongest tissue-specific cassette will be evaluated for long-term expression of therapeutic levels of microdystrophin in the mdx mouse model for DMD.

Gene Therapy Approaches for Intervertebral Disc Degeneration

Review of the literature concerning the development of gene therapy approaches for the treatment of intervertebral disc degeneration. To provide an overview of gene therapy principles, summarize the results of completed gene therapy studies, and discuss considerations for the direction of future research. Intervertebral disc degeneration is characterized by progressive loss of the proteoglycan matrix in the nucleus pulposus. Exogenous growth factors have been shown to transiently increase matrix synthesis. Gene therapy offers exciting potential to induce and sustain endogenous production of growth factors within the intervertebral disc and thus possibly alter the degenerative course. Published and presented scientific literature was examined. Several in vitro and in vivo studies have documented the capacity of gene therapy to favorably modify the biologic functions of intervertebral disc cells with the delivery of the cDNA for various growth factors. Currently, investigators are exploring the efficacy and safety of gene therapy in animal models of degeneration. With promising initial results and an immense potential clinical impact, gene therapy approaches for treatment of intervertebral disc degeneration will continue to receive dedicated research efforts.

Durable and stratum-specific gene expression in epidermis.

A number of genetic disorders are manifested in cutaneous epithelium and gene therapy approaches for treatment of such diseases are being considered. A successful gene therapy protocol requires durable and correctly targeted gene expression within the tissue. The continuous renewal and high levels of compartmentalization in epidermis are two challenges for a successful gene therapy of skin disorders. For those disorders which affect the upper layers of epidermis, vectors must be available that target stem cells, but remain silent until the progeny of these cells undergo differentiation. To explore the potential of long-term and targeted vector expression in epidermis, a hybrid retroviral vector encoding the reporter enhanced green fluorescent protein (EGFP) was constructed. The viral enhancer in the long terminal repeat of the vector was replaced with a 510-bp enhancer element of the human involucrin promoter. Keratinocyte-specific expression directed by the hybrid vector was demonstrated in culture and suprabasal-specific expression was observed in organotypic human epidermal cultures. In vivo transduction of mouse skin with this hybrid vector indicated long-term and stratum-specific expression of the transgene in mouse epidermis. The design of similar vectors for various gene therapy applications constitutes an important step toward clinically effective gene therapy.

CELLUAR PATHOPHYSIOLOGY OF PORTAL HYPERTENSION AND PROSPECTS FOR MANAGEMENT WITH GENE THERAPY

In summary, regulation of sinusoidal blood flow in normal and injured liver involves structural, cellular, and humoral components. Available data suggest that stellate cells, resident perisinusoidal mesenchymal cells with a histologic orientation in the sinusoid analogous to [figure: see text] vasoregulatory pericytes, modulate sinusoidal blood flow. This regulation by stellate cells is most evident in the context of liver injury but may apply also to the normal liver. The endothelin and NO systems are important in modulating stellate cell contractility, and their degree of equilibrium is significant in determining the level of local intrahepatic resistance, especially in the injured liver. Manipulation of either or both of these systems is feasible and effective in experimental models. Such findings have obvious clinical implications and are expected to set the [figure: see text] stage for novel gene therapy approaches for treatment of patients with portal hypertension.

Co-expression of mutated and normal adrenoleukodystrophy protein reduces protein function: implications for gene therapy of X-linked adrenoleukodystrophy.

Inherited defects in the X-chromosomal adrenoleukodystrophy (ALD; ABCD1) gene are the genetic cause of the severe neurodegenerative disorder X-linked adrenoleukodystrophy (X-ALD). Biochemically the accumulation of very long-chain fatty acids, caused by impaired peroxisomal beta-oxidation, is the pathognomonic characteristic of the disease. Due to the X-chromosomal inheritance of X-ALD no data are available to clarify the question whether mutated adrenoleukodystrophy proteins (ALDPs) can negatively influence normal ALDP function. Here we show that restoration of beta-oxidation in X-ALD fibroblasts following transient transfection with normal ALD cDNA is more effective in ALDP-deficient fibroblasts compared with fibroblasts expressing normal amounts of mutated ALDP. Furthermore, we utilized the HeLa Tet-on system to construct a stable HeLa cell line expressing a constant level of endogenous ALDP and doxycycline-inducible levels of mutated ALDP. The induction was doxycycline dosage-dependent and the ALDP correctly localized. Interestingly, although mutated ALDP increased >6-fold in a dosage-dependent manner the total amount of ALDP (mutated and normal) remained approximately even as demonstrated by western blot and flow cytometric analyses. Thus, apparently mutated and normal ALDP compete for integration into a limited number of sites in the peroxisomal membrane. Consequently, increased amounts of mutated ALDP resulted in decreased peroxisomal beta-oxidation and accumulation of very long-chain fatty acids. These findings have direct implications on future gene therapy approaches for treatment of X-ALD, since in some patients a non-functional endogenous protein could act in a dominant negative way or displace the introduced, normal protein.

Restoration of lymphocyte function in Janus kinase 3-deficient mice by retroviral-mediated gene transfer.

Janus kinase-3 (JAK3) deficiency has recently been identified as a cause of severe combined immunodeficiency (SCID) in humans. We used a mouse model of Jak3-deficient SCID to test a gene therapy approach for treatment of this disease. Transfer of a Jak3 retroviral vector to repopulating hematopoietic stem cells resulted in increased numbers of T and B lymphocytes, reversal of hypogammaglobulinemia, restoration of T-cell activation upon stimulation with mitogens, and development of an antigen-specific immune response after immunization. Analysis for vector copy number in lymphoid and myeloid populations showed a large in vivo selective advantage for Jak3-expressing lymphoid cells. These results show that gene replacement is a feasible treatment strategy for this disease and that naturally occurring in vivo selection of corrected cells is an important advantage of this approach.

Targeted vectors for gene therapy of cancer and retroviral infections.

Gene therapy has developed to a technology which rapidly moved from the laboratory bench to the bedside in the clinic. This implies safe, efficient and targeted gene transfer systems for suitable application to the patient. Beside the development of such gene transfer vectors of viral or nonviral origin, improvement of cell type specific and inducible gene expression is pivotal for successful gene therapy leading to targeted gene action. Numerous gene therapy approaches for treatment of cancer and retroviral infections utilize cell type specific and/or regulatable promoter and enhancer sequences for the selective expression of therapeutic genes in the desired cell populations and tissues. In this article the recent developments and the potential of expression targeting are reviewed for gene therapy approaches of cancer and retroviral infections.