Previous work established retinal expression of channelrhodopsin-2 (ChR2), an algal cation channel gated by light, restored physiological and behavioral visual responses in otherwise blind rd1 mice. However, a viable ChR2-based human therapy must meet several key criteria: (i) ChR2 expression must be targeted, robust, and long-term, (ii) ChR2 must provide long-term and continuous therapeutic efficacy, and (iii) both viral vector delivery and ChR2 expression must be safe. Here, we demonstrate the development of a clinically relevant therapy for late stage retinal degeneration using ChR2. We achieved specific and stable expression of ChR2 in ON bipolar cells using a recombinant adeno-associated viral vector (rAAV) packaged in a tyrosine-mutated capsid. Targeted expression led to ChR2-driven electrophysiological ON responses in postsynaptic retinal ganglion cells and significant improvement in visually guided behavior for multiple models of blindness up to 10 months postinjection. Light levels to elicit visually guided behavioral responses were within the physiological range of cone photoreceptors. Finally, chronic ChR2 expression was nontoxic, with transgene biodistribution limited to the eye. No measurable immune or inflammatory response was observed following intraocular vector administration. Together, these data indicate that virally delivered ChR2 can provide a viable and efficacious clinical therapy for photoreceptor disease-related blindness.

Studies of cell population dynamics and microenvironmental organization of B lymphopoiesis in the bone marrow of normal mice and in various genetically modified states have shown that cell loss, involving processes of apoptosis and macrophage-mediated cell deletion, is a prominent feature of the primary genesis of B lymphocytes. Balanced against the influence of proliferative stimulants, the programmed death of precursor B cells provides a quantitative control, determining the magnitude of the final output of functional B lymphocytes to the peripheral immune system. The cell loss mechanisms can be readily set in motion by external or systemic influences, making the B-cell output particularly vulnerable to suppression by ionizing irradiation, stress or other systemic mediators. In addition, however, cell loss exerts an important quality control in the formation of the primary B-cell repertoire. The combination of apoptosis and macrophage-mediated deletion, acting at successive stages of B-cell differentiation, efficiently eliminates many precursors having non-productive Ig gene rearrangements, cell cycle dysregulations, and certain autoreactive Ig specificities. Outstanding areas of further work abound. Important questions concern the nature of mechanisms which underlie the processes of B-cell apoptosis and macrophage deletion in bone marrow, the microenvironmental signals involved in B-cell life or death decisions and genetic factors which may override these B-cell culling mechanisms. The answers will be relevant to problems of autoimmune disease, humoral immunodeficiency and B-cell neoplasia.

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