AAV2 vector optimization for retinal ganglion cell-targeted delivery of therapeutic genes.

Recombinant adeno-associated virus (AAV)-2 has significant potential as a delivery vehicle of therapeutic genes to retinal ganglion cells (RGCs), which are key interventional targets in optic neuropathies. Here we show that when injected intravitreally, AAV2 engineered with a reporter gene driven by cytomegalovirus (CMV) enhancer and chicken β-actin (CBA) promoters, displays ubiquitous and high RGC expression, similar to its synthetic derivative AAV8BP2. A novel AAV2 vector combining the promoter of the human RGC-selective γ-synuclein (hSNCG) gene and woodchuck hepatitis post-transcriptional regulatory element (WPRE) inserted upstream and downstream of a reporter gene, respectively, induces widespread transduction and strong transgene expression in RGCs.

Cell-Specific Expression of Human SIRT1 by Gene Therapy Reduces Retinal Ganglion Cell Loss Induced by Elevated Intraocular Pressure.

SIRT1 prevents retinal ganglion cell (RGC) loss in several acute and subacute optic neuropathy models following pharmacologic activation or genetic overexpression. We hypothesized that adeno-associated virus (AAV)-mediated overexpression of SIRT1 in RGCs in a chronic ocular hypertension model can reduce RGC loss, thereby preserving visual function by sustained therapeutic effect. A control vector AAV-eGFP and therapeutic vector AAV-SIRT1 were constructed and optimized for transduction efficiency.

Selective Upregulation of SIRT1 Expression in Retinal Ganglion Cells by AAV-Mediated Gene Delivery Increases Neuronal Cell Survival and Alleviates Axon Demyelination Associated with Optic Neuritis.

Optic neuritis (ON), the most common ocular manifestation of multiple sclerosis, is an autoimmune inflammatory demyelinating disease also characterized by degeneration of retinal ganglion cells (RGCs) and their axons, which commonly leads to visual impairment despite attempted treatments. Although ON disease etiology is not known, changes in the redox system and exacerbated optic nerve inflammation play a major role in the pathogenesis of the disease. Silent information regulator 1 (sirtuin-1/SIRT1) is a ubiquitously expressed NAD-dependent deacetylase, which functions to reduce/prevent both oxidative stress and inflammation in various tissues.

Epigenetic changes during disease progression in a murine model of human chronic lymphocytic leukemia.

Epigenetic alterations, including gain or loss of DNA methylation, are a hallmark of nearly every malignancy. Changes in DNA methylation can impact expression of cancer-related genes including apoptosis regulators and tumor suppressors. Because such epigenetic changes are reversible, they are being aggressively investigated as potential therapeutic targets.

Synthetic peptide vaccine targeting a cryptic neutralizing epitope in domain 2 of Bacillus anthracis protective antigen.

Current evidence suggests that protective antigen (PA)-based anthrax vaccines may elicit a narrow neutralizing antibody repertoire, and this may represent a vulnerability with PA-based vaccines. In an effort to identify neutralizing specificities which may complement those prevalent in PA antiserum, we evaluated whether sequences within the 2beta2-2beta3 loop of PA, which are apparent in the crystal structure of heptameric but not monomeric PA, might represent a target for an epitope-specific vaccine for anthrax and, further, whether antibodies to these sequences are induced in rabbits immunized with monomeric PA. We evaluated the immunogenicity in rabbits of a multiple antigenic peptide (MAP) displaying copies of amino acids (aa) 305 to 319 of this region.

Genetic vaccines for anthrax based on recombinant adeno-associated virus vectors.

Bacillus anthracis represents a formidable bioterrorism and biowarfare threat for which new vaccines are needed with improved safety and efficacy over current options. Toward this end, we created recombinant adeno-associated virus type 1 (rAAV1) vectors containing synthetic genes derived from the protective antigen (PA) or lethal factor (LF) of anthrax lethal toxin (LeTx) and tested them for immunogenicity and induction of toxin-neutralizing antibodies in rabbits. Codon-optimized segments encoding activated PA (PA63), or LF, were synthesized and cloned into optimized rAAV1 vectors containing a human cytomegalovirus (hCMV) promoter and synthetic optimized leader.

CpG island methylation and expression of the secreted frizzled-related protein gene family in chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (CLL) is characterized by a clonal accumulation of mature neoplastic B cells indicating disruption of apoptosis. Restriction Landmark Genome Scanning was done to identify novel target genes silenced by CpG island methylation in CLL. Secreted frizzled-related protein 4 (SFRP4), a negative regulator of the Wnt signaling pathway, was found to be frequently methylated in CLL samples.

Global assessment of promoter methylation in a mouse model of cancer identifies ID4 as a putative tumor-suppressor gene in human leukemia.

DNA methylation is associated with malignant transformation, but limitations imposed by genetic variability, tumor heterogeneity, availability of paired normal tissues and methodologies for global assessment of DNA methylation have limited progress in understanding the extent of epigenetic events in the initiation and progression of human cancer and in identifying genes that undergo methylation during cancer. We developed a mouse model of T/natural killer acute lymphoblastic leukemia that is always preceded by polyclonal lymphocyte expansion to determine how aberrant promoter DNA methylation and consequent gene silencing might be contributing to leukemic transformation. We used restriction landmark genomic scanning with this mouse model of preleukemia reproducibly progressing to leukemia to show that specific genomic methylation is associated with only the leukemic phase and is not random.

Epigenetic profiling in chronic lymphocytic leukemia reveals novel methylation targets.

CpG island methylation is an epigenetic alteration that contributes to tumorigenesis by transcriptional inactivation of genes. Little is known about the overall levels of CpG island methylation in chronic lymphocytic leukemia (CLL). To provide a baseline estimate of global aberrant methylation and identify target sequences for additional investigation, we performed Restriction Landmark Genomic Scanning on 10 CLL samples.

Transcription of the Drosophila CKI gene dacapo is regulated by a modular array of cis-regulatory sequences.

Organogenesis requires coordination between developmental programs and cell cycle progression. The Cip/Kip families of cyclin-dependent kinase inhibitor (CKI) are main effectors linking these two programs. In many instances, expression of Cip/Kip CKIs are increased abruptly in cells entering their last mitotic cycle, suggesting that CKI expression is developmentally regulated.