AAV-mediated subcellular targeting of heterologous rhodopsins in retinal ganglion cells
Inventors
Assignees
Publication Number
US-12343408-B2
Publication Date
2025-07-01
Expiration Date
2031-05-04
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Abstract
Microbial type rhodopsins, such as the light-gated cation-selective membrane channel, channelrhodopsin-2 (Chop2/ChR2) or the ion pump halorhodopsin (HaloR) are expressed in retinal ganglion cells upon transduction using recombinant AAV vectors. Selective targeting of these transgenes for expression in discrete subcellular regions or sites is achieved by including a sorting motif in the vector that can target either the central area or surround (off-center) area of these cells. Nucleic acid molecules comprising nucleotide sequences encoding such rhodopsins and sorting motifs and their use in methods of differential expression of the transgene are disclosed. These compositions and methods provide significant improvements for restoring visual perception and various aspects of vision, particular in patients with retinal disease.
Core Innovation
The invention relates to the targeted expression of microbial-type rhodopsins, such as channelrhodopsin-2 (ChR2) and halorhodopsin (HaloR), in retinal ganglion cells (RGCs) using recombinant adeno-associated virus (AAV) vectors. By including specific peptide or polypeptide sorting motifs within the nucleic acid vectors, selective expression of these rhodopsins is achieved in discrete subcellular regions of retinal neurons. This differential targeting, particularly to the center or surround (off-center) areas of RGCs, allows for improved spatial control and mimics the antagonistic center-surround receptive fields, which enhances visual spatial processing and contrast sensitivity.
The problem being addressed is the lack of effective treatments for inherited retinal degenerations that lead to blindness due to loss of photoreceptor cells. Existing therapies either focus on preserving remaining photoreceptors or replacing lost ones but face significant obstacles, including inefficacy in advanced stages and impracticality of transplantation or prosthetic devices. Prior work demonstrated expression of microbial rhodopsins in inner retinal neurons to restore some light sensitivity, but without spatial resolution or subcellular specificity, limiting the restored visual function.
The invention improves upon previous approaches by directing expression of light-sensitive rhodopsins to subcellular sites within retinal neurons, especially RGCs, through the use of sorting motifs linked in-frame to the rhodopsin coding sequences. This targeted expression spares axonal regions and restricts expression to somatodendritic or proximal dendritic regions, enhancing spatial resolution and visual contrast akin to natural retinal processing. The compositions include nucleic acid molecules comprising sequences encoding rhodopsins, sorting motifs, promoters such as the CAG promoter, optional reporter genes like GFP, and regulatory elements all packaged in recombinant AAV vectors constructed for stable, site-selective expression.
Claims Coverage
The patent includes multiple inventive features focused on nucleic acid molecules, vectors, and methods for targeted subcellular expression of rhodopsins in retinal neurons. The main inventive features encompass polynucleotide molecules with specified sorting motifs, recombinant AAV expression vectors, and corresponding methods of restoring light sensitivity by selective subcellular expression.
Polynucleotide molecules encoding rhodopsins with specific sorting motifs for subcellular targeting
The polynucleotide molecules comprise (a) a nucleotide sequence encoding a light-gated channel rhodopsin or a light-driven ion pump rhodopsin and (b) a linked nucleotide sequence encoding a sorting motif selected from nicotinic acetylcholine receptor a7 subunit (nAchR), voltage-gated potassium channel 4.2 (Kv4.2), telencephalin (TLCN), and AMPA receptor GluR1 subunit. The molecules also include operatively linked promoter sequences and polyadenylation sequences.
Use of promoters and regulatory sequences to enhance expression
The promoter sequence is cytomegalovirus enhancer/chicken β-actin promoter (CAG), with the polyadenylation sequence chosen from bovine growth hormone (bGHpolyA) or SV40-derived polyadenylation sequences. Optionally, the transcriptional regulatory sequence includes woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Multiplex constructs can include reporter polypeptide sequences linked in frame.
Recombinant AAV2 expression vectors with targeted rhodopsin expression
The recombinant AAV2 vectors incorporate the polynucleotide molecules with rhodopsin and sorting motifs flanked by AAV2 inverted terminal repeats (ITRs). The vectors preserve the promoter and regulatory elements enabling expression in selective subcellular retinal neuron regions. Exemplary vector structures with or without green fluorescent protein (GFP) are detailed.
Methods of restoring light sensitivity by subcellular targeted expression
Methods involve delivering the polynucleotide molecule or rAAV2 vector to retinal neurons, preferably RGCs, and expressing rhodopsins in selected subcellular regions determined by the sorting motifs to restore light sensitivity. Selectivity is achieved by the sorting motifs targeting center or surround receptive field regions.
The claims collectively cover compositions comprising nucleic acid molecules and recombinant viral vectors encoding light-sensitive rhodopsins fused to sorting motifs that enable subcellular targeting within retinal neurons. The invention also incorporates promoters and regulatory sequences to optimize expression, and methods of delivering these constructs to restore or adjust light sensitivity in retinal cells with subcellular precision, improving vision restoration approaches.
Stated Advantages
The compositions and methods enhance spatial control and specificity of rhodopsin expression in retinal ganglion cells, leading to improved contrast and spatial resolution in restored vision.
The invention enables restoration of light sensitivity by mimicking the physiological center-surround receptive fields of retinal neurons, allowing signals closer to those from healthy retinas to be transmitted to higher visual centers.
The approach avoids introducing exogenous cells, tissues, or physical devices, circumventing obstacles faced by transplantation or prosthetic methods.
The targeted expression reduces unwanted expression in axonal regions, potentially improving functional outcomes and reducing side effects.
The use of microbial rhodopsins that bind all-trans retinal avoids the need for vertebrate visual cycle components, simplifying the therapeutic strategy.
Documented Applications
Restoring visual perception and various aspects of vision in patients with retinal degenerative diseases, including inherited blinding disorders such as retinitis pigmentosa and age-related macular degeneration.
Targeted gene therapy to retinal ganglion cells or bipolar cells in retinas with photoreceptor loss to reinstate light sensitivity and visual signal transmission.
Methods for improving contrast sensitivity and spatial resolution of restored vision by differential subcellular expression of light-sensitive ion channels.
Potential combination with visual prostheses and devices to enhance vision restoration.
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