April 11, 2011

SENS 2010 research report

SENS has a research report for November, 2010

SENS Foundation conducts intramural research in its Research Center in Mountain View, California. The primary focus of our intramural work is LysoSENS –
investigating novel lysosomal hydrolases against intracellular aggregates that impair
cell function – and we recently produced a detailed and comprehensive LysoSENS
planning document in collaboration with our extramural project at Rice University.

We have also arranged for research in the MitoSENS strand – obviating
mitochondrial DNA deletions – to be conducted at the Research Center, following
the negotiation of a transfer agreement with Dr Corral-Debrinski covering materials
produced, and used in, previous successful work by her group. Dr Matthew “Oki”
O'Connor joined us in September to initiate this project.



Lysosens

2010 milestones. The creation of an internal A2E synthesis capability enabled efficient target enzyme evaluation. Our successful development of a protein purification protocol resulted in the isolation of an active, A2E-degrading enzyme.

The year ahead. We will express and purify additional candidate enzymes for both macular degeneration and atherosclerosis projects; establish activity of these additional enzymes in vitro; perform cell-uptake studies, in RPE cells for A2E and in macrophages for 7-ketocholesterol (7KC); and perform initial toxicity studies.

One of our priorities has been to overcome the limitations on our ability to test potential A2Edegrading enzymes, caused by a restricted supply of the target molecule. We have now successfully synthesized A2E, purifying it by flash chromatography and HPLC, and confirming it to be spectrophotometrically identical to that produced in Janet Sparrow's laboratory. Our ability to produce this material ‘in house’ is a key element in the expansion of our screening and evaluation of existing and newly-purified A2E-degrading enzymes.

Rice University Lysosens
Location. Rice University, Houston, Texas

2010 milestones. We have designed and synthesised modified versions of
eight microbial hydrolases, the expressions of which are upregulated by 7KC;
developed an HPLC assay; and expressed proteins for target candidates.

The year ahead. We will develop further expression testing for cloned proteins
and conduct in vitro activation testing.

Mitosens

2010 milestones. We have established an in-house MitoSENS program, based
on previous, funded work in the INSERM laboratory of Dr Corral-Debrinski.
The year ahead. We will transiently express mitochondrially-encoded proteins
(ND1, ND4, ATP6) from provided expression plasmids; confirm localization to
mitochondria by immunofluorescence; design and synthesize “nuclear”
cytochrome B followed by similar expression and localization; demonstrate
Complex III functional rescue with nuclear expression of cytochrome B.

ApoptoSENS and RepleniSENS

Location. University of Arizona, Tucson, Arizona
2010 milestones. We have constructed a prototype T-cell “scrubber” and confirmed that it reduces the abundance of dysfunctional immune cells; prepared suitably infected mice and verified their immune status; and applied permutations of immunorejuvenation therapies.

The year ahead. We will analyse the data collected and publish results.

The project seeks to test whether removal of accumulated age- and virus-related T-cell clonal expansions and/or T-cell repertoire rebalancing (i) restores functionality of T-cell compartment in the aged organism, and (ii) improves immune defense against new infection. This series of longitudinal experiments was designed using 2 mouse models of persistent viral infection known to result in the development of virus-specific CD8+ T-cell expansions (TCE). Such expansions are thought to “crowd out” the naïve T-cells needed to target novel pathogens, limiting the ability of the immune system. These experiments will directly test methods for the removal and/or neutralization of virally-induced expansions in aging mice, and the ability of these methods to rebalance and rejuvenate the aging immune system to restoring youthful, robust immune defense. The study began at the end of 2008 and will conclude in June 2011. At the beginning of the study, 180 mice were received and sorted into five groups for treatment and control. Mice were infected
with HSV-1 or MCMV, or were not infected. Evaluation of the virus-specific CD8 T-cell populations in all infected mice showed evidence of the initial inflation of those populations within aging mice, particularly within the MCMV-infected groups. At 55 weeks, the dual expression of the cell surface markers KLRG1 and PD-1 by virus-specific CD8 T-cells (an indication of functionally “exhausted” cells) was beginning to emerge, particularly within MCMV-infected groups. In late May and June 2010, additional therapeutic interventions were performed

OncoSENS

Location. Albert Einstein College of Medicine, Bronx, New York

2010 milestones. We developed a method to determine gene-specific DNA
methylation patterns in single cells; tested this protocol for gene-specific DNA
methylation patterns in different types of single cell; and optimized the
protocol to isolate the nuclei of single neurons.

The year ahead. We will develop a novel method for single-cell global gene
methylation analysis and study cell-to-cell variation in DNA methylation
patterns among neurons during mouse aging.

ApoptoSENS

Location. Buck Institute for Age Research, Novato, California

2010 milestones. Work at the Foundation’s Research Center identified senescence markers and toxins. We have engineered transiently active suicide genes.

The year ahead. We will engineer permanently active suicide genes; determine their effect on the abundance of senescent cells; and determine further, downstream effects.

AmyloSENS

Location. University College Dublin, Dublin, Ireland
University of Texas, Houston, Texas
2010 milestones. This is a new project for the Foundation.

The year ahead. We will identify antibodies and fragments that bind to, an catalytically cleave, transthyretin fibrils and high-copy-number oligomers; and
identify the structure and composition of other amyloids and their role in age related diseases.


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