Viral capsid protein, viral particle, and use of adeno-associated viruses variants.

Abstract

The present invention provides variants of aav that exhibit a preference for retrograde movement in neurons and methods for utilizing such variants.

Description

1 / 53 Viral capsid protein, viral particle, and use of adeno-associated viruses variants. Federally Supported Research or Development

[0001] The present invention was made with government support under EY022975 granted by the National Institutes of Health. The government has certain rights in the invention. Cross-referencing to related patent applications.

[0002] This patent application claims the benefit of priority under 35 USC § 119(e) to US Patent Application No. 62 / 350 361 filed June 15, 2016, and US Patent Application No. 62 / 404 585 filed October 5, 2016. Technical Field

[0003] This disclosure refers generally to adeno-associated viruses variants and methods of manufacture and use. Background

[0004] Brain functions such as perception, cognition, and movement control depend on the coordinated action of large-scale neural networks, which are composed of local circuit modules that perform specific computations and are linked by long-range connections that distribute the results of these computations. Such long-range connections are formed by specialized projection neurons that often include several mixed classes, each projecting to a different downstream target within the network. Projection neurons have also been implicated in the spread of various neurodegenerative diseases from spatially localized sites of onset. Thus, the ability to selectively target certain classes of projection neurons for transgene release (e.g., for activity monitoring / manipulation or genome editing for targeted gene knockouts or repair of pathological mutations) will be important for gaining insight. Petition 870260060163, dated 06 / 19 / 2026, p. 7 / 128 2 / 53 studies on how large-scale networks contribute to brain function and, in the long run, to therapeutic intervention in neurodegenerative diseases.

[0005] Viral vectors constitute an important class of tools for introducing transgenes into specific neuronal populations, and are by far the best option for genetic access to target projection neurons through entry into axonal terminals and retrograde transport of their payload to cell nuclei. A number of naturally evolved neurotrophic viruses exhibit retrograde dispersal as part of their life cycle, including rabies, polio virus, and herpes simplex virus (HSV), among others. Of these, rabies virus is particularly neuroinvasive and spreads rapidly through the nervous system via transcellular transfer. However, its potential for both biological research and gene therapy is hampered by excessive virulence, although progress is being made towards reducing its toxicity.In addition to naturally neurotrophic lineages, many other viruses can infect neurons when administered directly to the nervous system, with pseudorabies (SuHV1, actually a herpesvirus), adenovirus, and lentivirus most commonly used in animal research. Canine adenovirus-2 (CAV-2) shows the best infectivity and retrograde transport in this class of viruses and has increasingly become the reagent of choice for accessing projection neurons. CAV-2, however, mediates only modest levels of transgene expression, shows potential for toxicity, and is currently not readily compatible with robust, scalable production for clinical-grade or even large-animal studies. Thus, the development of an easily manufactured, non-toxic viral vector that provides flexible packaging of different transgenes, is robustly internalized and retrogradely transported by axons, and supports expression is crucial. Petition 870260060163, dated 06 / 19 / 2026, page 8 / 128 3 / 53 of high-level, long-term cargo remains an urgent need. Summary

[0006] Efficient retrograde access to projection neurons for the delivery of sensors and effectors constitutes an important and enabling capability for circuit dissection. Such an approach may also be useful for gene therapy, including the treatment of neurodegenerative disorders characterized by pathological dissemination through highly distributed and functionally connected networks. Viral vectors, in particular, are powerful gene delivery vehicles to the nervous system, but all available tools suffer from inefficient retrograde transport or limited clinical potential. To address this need, targeted in vivo evolution was applied to engineer potent retrograde functionality in the capsid of adeno-associated virus (AAV) – a vector that has shown promise in neuroscience and clinical research.The variant described here, referred to as retro-rAAV2, allows robust retrograde access to projection neurons with efficiency comparable to classic synthetic retrograde labeling reagents, and enables high levels of expression sufficient for functional circuit interrogation and in vivo genome editing using CRISPR / Cas9 in targeted neuronal populations.

[0007] In one aspect, a viral capsid protein is provided that includes a selected sequence from the group consisting of xxDxTKx (SEQ ID NO: 1) and xDxTKxx (SEQ ID NO: 2). In one embodiment, the viral capsid protein has at least 95% sequence identity to SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or 78. In another embodiment, the viral capsid protein has the sequence shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, Petition 870260060163, dated 06 / 19 / 2026, page 9 / 128 4 / 53 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, or 78. In one embodiment, the viral capsid protein is encoded by a nucleic acid having at least 95% sequence identity to SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, or 77. In one embodiment, the viral capsid protein is encoded by a nucleic acid having the sequence shown in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, and 77.

[0008] In yet another aspect, a viral particle is provided that includes a viral capsid protein as described herein. In some embodiments, the viral particle exhibits a preference for retrograde movement. In some embodiments, the viral particle has retrograde transport capability.

[0009] In some embodiments, a viral particle as described herein further includes a nucleic acid encoding a payload. In some embodiments, the nucleic acid encoding a payload includes a promoter sequence operably linked to a coding sequence that encodes the payload. Representative promoter sequences include, without limitation, synapsin-1, CMV, GFAP, CAG, CaMKII, MBP, EF1alpha, TER, and mDIx. In some embodiments, the coding sequence encoding the payload is selected from the group consisting of a protein-coding gene and an inhibitory RNA nucleic acid. In some embodiments, the inhibitory RNA nucleic acid is an antisense oligonucleotide, an siRNA, or an RNAi.

[0010] In some embodiments, the payload is an effector protein. Representative effector proteins include, but are not limited to, a recombinase (e.g., Cre or Flp), a gene editing system (e.g., CRISPR / Cas9, TALEN, zinc finger nuclease), rea Petition 870260060163, dated 06 / 19 / 2026, page 10 / 128 5 / 53 optogenetic agents (activators (e.g., channel rhodopsin or its variants) or inhibitors (e.g., halorhodopsin or Arch)), chemogenetic reagents (e.g., activator / inhibitor versions of the DREADD or PSAM / PSEM systems), activators and / or inhibitors of cell system pathways, and enzymes for epigenetic control.

[0011] In some embodiments, the payload is an optical reporter construct. Representative optical reporter constructs include, without limitation, GCaMP6 (s, m, or f), a fluorophore (e.g., green fluorescent protein (GFP), enhanced GFP (EGFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), a color motion construct (e.g., a payload expressing one reporter in one cell population and a different reporter in another population), glucose sensors, jRCaMP, jRGECO, and CaMPARI, voltage indicators, secondary messengers, signaling receptors, transcriptional reporters, epigenetic reporters, and neuromodulatory reporters.

[0012] In some forms, the payload is a viral protein. A representative viral protein is the rabies G protein. In some embodiments, the viral protein is a protein that complements the function of another virus other than AAV or a protein that relates to cellular and transcellular transport.

[0013] In some embodiments, the coding sequence encoding the payload is a therapeutic gene. In some embodiments, the therapeutic gene is for the treatment of a neurodegenerative disorder. In some embodiments, the therapeutic gene is HSP104 for the treatment of Alzheimer's disease or other diseases with toxic protein aggregates. In some embodiments, the therapeutic gene is frataxin for the treatment of Friedreich's ataxia. In some embodiments, the therapeutic gene is lysosomal glucocerebrosidase (GBA) for Petition 870260060163, dated 06 / 19 / 2026, page 11 / 128 6 / 53 treatment of Parkinson's disease. In some modalities, the therapeutic gene is polyQ-binding protein for the treatment of Huntington's disease. In some modalities, the therapeutic gene is survival motor neuron 1 for the treatment of spinal muscular atrophy, amyotrophic lateral sclerosis (ALS), autism, dementia, peripheral neuropathy, schizophrenia, or retinal degeneration.

[0014] In some modalities, the payload is a therapeutic fraction. A representative therapeutic fraction is an antibody or its fragment. A representative therapeutic fraction is an immunomodulatory protein. A representative therapeutic fraction is an RNA interference molecule.

[0015] In some embodiments, the viral particles described here exhibit retrograde access up to two orders of magnitude greater to corticospinal projection neurons than canine adenovirus-2 (CAV-2). In some embodiments, retrograde access to corticospinal projection neurons or afferents to the dorsomedial striatum (DMS) is comparable to the synthetic tracer, Fluoro-Gold fluorescent beads.

[0016] In another aspect, a process of releasing a payload to one or more neurons is provided. Such a process typically involves contact of one or more neurons with an adeno-associated virus variant (AAV) that includes a payload packaged therein, where the AAV variant includes a capsid protein comprising a sequence selected from the group consisting of xxDxTKx (SEQ ID NO: 1) and xDxTKxx (SEQ ID NO: 2). In some embodiments, the AAV variant exhibits retrograde movement in neurons.

[0017] In some modalities, the neurons are projection neurons. In some modalities, the neurons are in a subject (e.g., in the central nervous system (CNS) in a subject). In some modalities, the subject is a human. In some modalities Petition 870260060163, dated 06 / 19 / 2026, page 12 / 128 7 / 53 des, the subject is a non-human (e.g., a primate, a rodent, a reptile, or a bird). In some embodiments, the contact step is in cell culture. In some embodiments, the contact step is in vivo via intracranial, intraspinal, or intramuscular injection.

[0018] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the processes and compositions of matter pertain. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the processes and compositions of matter, appropriate methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. Description of Drawings

[0019] Figure 1 shows the directed evolution of retro-rAAV2. Panel A of Figure 1 is a schematic view of the targeted evolution procedure. Plasmid libraries containing variant AAV cover genes previously generated through error-prone PCR, peptide insertion, loop region randomization, and DNA shuffling were packaged and injected into substantia nigra or deep cerebellar nuclei. Three weeks later, striatal or metencephalon tissue, respectively, was removed, viral genomes isolated, and selected cover genes amplified and packaged for the next round of selection. Panel B of Figure 1 shows the mixed subpopulations of cortical neurons projecting to the striatum (CP: caudate-putamen), thalamus, and superior colliculus (SC) retrogradely labeled by injection of retro-rAAV2 carrying different fluorescent proteins into the corresponding camp. 8 / 53 po axônial.

[0020] Figure 2 shows results from experiments to quantify retrograde transport efficiency. Panel A of Figure 2 shows the corticopontine tract labeled via basal tip injection of retrorAAV2 [Top panel of Panel A shows a schematic view of the experiment; targeting consistency and injection quality were monitored via co-injection of AAV1-CAG-EGFP. Bottom panel of Panel A shows the expression level 3 weeks after injection. Scale bar: 1 mm]. Panel B of Figure 2 shows the quantification assay design [Top panel of Panel B shows a schematic view of the experiment; arrow indicates expected nuclear GFP labeling in cortical neurons of the corticopontine tract. Middle panel of Panel B shows a representative image of a brain injected with retro-rAAV2. Scale bar: 1 mm]. Panel C of Figure 2 shows a schematic view of the semi-automated quantification procedure.Fluorescent nuclei (green) were automatically detected and counted along a manually drawn line tracing the length of cortical layer V (black). Panel D of Figure 2 shows the retrograde transport efficiency for different AAV serotypes and for canine adenovirus-2 (CAV-2). Error bars represent SEM. See also Figure 8.

[0021] Figure 3 shows results from experiments to demonstrate the generality of retrograde transport provided by retro-rAAV2. Panel A of Figure 3 are representative images showing extensive labeling in the main input structures to the dorsal striatum including cortex (image #1), amygdala (image #3), and thalamus (image #4). Panel B of Figure 3 shows a schematic view of automated whole-brain quantification of retrograde labeling. Rosa26-LSL-H2B-GFP brains injected with Cre-hSyn1 retrorAAV2 were imaged for visualization of stained nuclei. Petition 870260060163, dated 06 / 19 / 2026, page 14 / 128 9 / 53 DAPI and green fluorescence of nuclei expressing H2B-GFP. The green channel is used to detect labeled neurons; the blue channel is aligned to standard mouse brain Nissl images from the Allen Brain Institute. The alignment allows detected neurons to be assigned to different regions using the annotation provided by the brain atlas. Scale bar: 1.25 mm. Panel C of Figure 3 shows whole-brain quantification of retrograde labeling outside a small region of the dorsomedial striatum. Abbreviations for the different brain areas are given according to the Allen Brain Atlas. Arrow highlights SNc. Error bars represent SEM.

[0022] Figure 4 shows data from experiments in which the retro-rAAV2 system was combined with Cre booster lines. Panel A of Figure 4 shows a schematic view of the experiment. RetrorAAV2 carrying a Cre-dependent color motion fluorescent reporter was injected into the V-line-specific Cre striatum of the cortical layer. Panel B of Figure 4 shows the two differentially labeled corticostriatal pathways through a Cre-dependent inversion of the reporter in one pathway but not the other.

[0023] Figure 5 shows data from experiments demonstrating that retro-rAAV2 supports sufficient transgene expression for functional circuit interrogation. Panel A of Figure 5 is a schematic view of the experiment. Calcium-indicating GCaMP6f expression is restricted to corticopontine neurons using localized injection of retro-rAAV2 into basal pons. Panel B of Figure 5 is a cross-section of the brain showing GCaMP6f expression throughout the corticopontine tract. Panel C of Figure 5 shows the maximum projection of a two-photon calcium image showing apical dendrites and V-layer pyramidal tract somas. Panel D of Figure 5 shows the activity of 89 ROIs during a single-handed strike repetition (dashed line represents the voltage siga signal). Panel E and Pf Petition 870260060163, dated 06 / 19 / 2026, page 15 / 128 10 / 53 nel F of Figure 5 are two examples of simple corticopontine neurons during 40 consecutive experiments (same animal as in Panel B and Panel D of Figure 5).

[0024] Figure 6 shows data demonstrating that the retro-rAAV2 system enables in vivo genome editing using CRISPR / Cas9. Panel A of Figure 6 is a schematic view of the experiment [Top panel of Panel A shows that the retro-rAAV2 system was used to deliver a single guide RNA combination - Staphylococcus aureus Cas9 engineered for tdTomato expression ablation. Bottom panel of Panel A shows that retro-rAAV2 carrying the SaCa9 anti-tdTomato payload was injected into the basal bridges of mice expressing tdTomato from a single genomic locus in layer V neurons]. Panel B of Figure 6 shows representative images of brain sections from animals that received the CRISPR / Cas9 system targeted against tdTomato or carrying an untargeted guide. SaCa9 was epitope-tagged, allowing identification of retrogradely labeled neurons (green channel).Upward arrows show the expected labeling following successful tdTomato ablation; downward arrows show the expected labeling if tdTomato expression is unchanged. Panel C of Figure 6 shows ablation efficiency. Error bars represent SEM.

[0025] Figure 7 shows data demonstrating that rAAV2-retro mediates efficient access to projection neurons in the rat (the data shown in Figure 7 are related to the data shown in Figure 1). Panel A of Figure 7 is a schematic view of the injection. Separate batches of eAAV2-retro expressing EGFP or tdTomato were injected into the striatum or superior colliculus, respectively. Panels B and E of Figure 7 show projection neurons in various brain regions accessed via these localized injections and imaged 3 weeks following virus release. Petition 870260060163, dated 06 / 19 / 2026, page 16 / 128 11 / 53

[0026] Figure 8 shows representative images of animal brains injected with various AAV serotypes in the basal pons, showing that no naturally occurring AAV serotype matches the performance of retro-rAAV2 in the corticopontine circuit (the data shown in Figure 8 are related to the data shown in Figure 2).

[0027] Figure 9 shows data illustrating reduced heparin affinity for retro-rAAV2 compared to its parent serotype AAV2 (the data shown in Figure 9 are related to the data shown in Figure 1). Panel A of Figure 9 is a schematic view of the heparin binding assay. Panel B of Figure 9 shows a virus fraction eluted with increasing NaCl concentrations following loading in 150 mM NaCl. The loading fraction represents the viruses recovered in the flow-through column after sample loading in 150 mM NaCl. Error bars represent the standard deviation. Detailed Description

[0028] Recombinant adeno-associated viruses (rAAVs) have emerged as an effective platform for in vivo gene therapy, as they mediate high levels of gene expression, are non-toxic, and evoke minimal immune responses. These properties were central to the decision to grant the first full regulatory approval of any rAAV-mediated gene therapy treatment for restoration of lipoprotein lipase deficiency (see, for example, Gaudet et al., 2010, Atherosclerosis Supplem., 11:55-60). rAAVs hold great promise in clinical trials for a range of neurological disorders, and they constitute some of the most widespread vectors in neuroscience research. Since the original discovery that AAVs can undergo retrograde transport (Kaspar et al., 2002, Mol. Ther., 5:50-56), rAAVs have provided some degree of retrograde access for projection neurons in selective circuits. Petition 870260060163, dated 06 / 19 / 2026, page 17 / 128 12 / 53 of them, but their natural tendency for retrograde transport is low, hindering efforts to address the role of projection neurons in circuit computations or disease progression.

[0029] This disclosure describes a novel rAAV variant (rAAV2retro) that, in addition to its regular ability to infect neuronal cell bodies at the exposure site, is robustly internalized by axons and mediates retrograde access to projection neurons with efficiency comparable to classical retrograde labeling reagents such as synthetic dyes. The rAAV2-retro gene delivery system described herein can be used on its own or in conjunction with Cre recombinase trigger lines to achieve high-level, long-term transgene expression that is sufficient for effective functional interrogation of neural circuit function, as well as for genome editing in target neuronal populations.

[0030] Retrograde transport propels molecules and / or organelles away from axon terminals toward the cell body. Retrograde axonal transport is mediated by cytoplasmic dynein and is used, for example, to send chemical messages and endocytosis products directed to endolysosomes from the axon back into the cell. Operating in vivo at average speeds of approximately 2 pm / s, rapid retrograde transport can cover 10–20 centimeters per day. Retrograde transport returns synaptic vesicles and other materials to the soma and informs the soma of conditions at the axon terminals. Thus, as used here, retrograde transport refers to movement along an axon toward its cell body. Polypeptide and nucleic acid sequences of adeno-associated viruses (AAVs)

[0031] As described herein, consensus sequences have been identified that, when present in an AAV capsid protein, result in a profound improvement in retrograde transport efficiency. Petition 870260060163, dated 06 / 19 / 2026, page 18 / 128 13 / 53 grade. Those consensus sequences are xxDxTKx (SEQ ID NO: 1) or xDxTKxx (SEQ ID NO: 2). To demonstrate the effectiveness of the consensus sequences described here in providing retrograde transport capability, seventeen different capsid sequences, each containing a consensus sequence as described here, were generated and shown to exhibit a preference for retrograde movement in neurons. The nucleic acid sequences of those 40 capsid sequences are shown in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, and 77, and the capsid polypeptides, each containing one of the consensus sequences described herein, are shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78, respectively.

[0032] In addition to the capsid polypeptides having the sequences shown in SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78, polypeptides are provided that have at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity) for the capsid polypeptides having the sequences shown in SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, and 78.Similarly, in addition to nucleic acid molecules having the sequences shown in SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, and 77, nucleic acid molecules are provided that have at least 95% sequence identity (e.g., at least 96%, at least 97%, at least 98%, at least 99%, or 100%). Petition 870260060163, dated 06 / 19 / 2026, p. 19 / 128 14 / 53 of sequence identity) for nucleic acid molecules having the sequences shown in SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, and 77.

[0033] In sequence identity percentage calculation, two sequences are aligned and the number of identical nucleotide or amino acid residue matches between the two sequences is determined. The number of pairings is divided by the length of the aligned region (i.e., the number of nucleotides or amino acid residues aligned) and multiplied by 100 to arrive at a sequence identity percentage value. It will be appreciated that the length of the aligned region can be a portion of one or both sequences up to the size of the entire length of the shorter sequence. It will also be appreciated that a single sequence can align with more than one other sequence and therefore may have different sequence identity percentage values ​​over each aligned region.

[0034] The alignment of two or more sequences to determine percentage sequence identity can be performed using the algorithm described by Altschul et al. (1997, Nucleic Acids Res., 25:3389 3402) as incorporated into BLAST programs (Basic Local Alignment Search Tool), available at ncbi.nlm.nih.gov on the World Wide Web. BLAST searches can be performed to determine the percentage sequence identity between a sequence (nucleic acid or amino acid) and any other sequence or portion thereof aligned using the Altschul et al. algorithm. BLASTN is the program used to align and compare the identity between nucleic acid sequences, while BLASTP is the program used to align and compare the identity between amino acid sequences. When using BLAST programs to calculate the percentage of Petition 870260060163, dated 06 / 19 / 2026, page 20 / 128 15 / 53 identity between one sequence and another sequence, the default parameters of the respective programs are generically used.

[0035] Vectors containing nucleic acid molecules encoding polypeptides are also provided. Vectors, including expression vectors, are commercially available or can be produced through recombinant technology. A vector containing a nucleic acid molecule may have one or more expression elements operably linked to such a nucleic acid molecule, and may also include sequences such as those encoding a selectable marker (e.g., an antibiotic resistance gene), and / or those that can be used in the purification of a polypeptide (e.g., 6xHis tag). Expression elements include nucleic acid sequences that direct and regulate the expression of nucleic acid-coding sequences. An example of an expression element is a promoter sequence.Expression elements may also include one or more introns, enhancer sequences, response elements, or inducible elements that modulate the expression of a nucleic acid molecule. Expression elements may be of bacterial, yeast, insect, mammalian, or viral origin, and vectors may contain a combination of expression elements from different origins. As used herein, operably linked means that expression elements are positioned in a vector relative to a coding sequence so as to direct or regulate the expression of the coding sequence.

[0036] A nucleic acid molecule, for example, a nucleic acid molecule in a vector (e.g., an expression vector, a viral vector) can be introduced into a host cell. The term host cell refers not only to the particular cell(s) into which the nucleic acid molecule has been introduced, but also to the progeny or potential progeny of such a cell. Many host cells Petition 870260060163, dated 06 / 19 / 2026, page 21 / 128 16 / 53 suitable host cells are known to those skilled in the art; host cells may be prokaryotic cells (e.g., E. coli) or eukaryotic cells (e.g., yeast cells, insect cells, plant cells, mammalian cells). Representative host cells may include, without limitation, A549, WEHI, 3T3, 10T1 / 2, BHK, MDCK, COS 1, COS 7, BSC 1, BSC 40, BMT 10, VERO, WI38, HeLa, 293 cells, Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast, hepatocytes and myoblast cells derived from mammals including humans, monkeys, mice, rats, rabbits and hamsters. Methods for introducing nucleic acid molecules into host cells are well known in the art and include, but are not limited to, calcium phosphate precipitation, electroporation, heat shock, lipofection, microinjection, and virus-mediated nucleic acid transfer (e.g., transduction).

[0037] With respect to polypeptides, purified refers to a polypeptide (i.e., a peptide or a polypeptide) that has been separated or purified from naturally occurring cellular components. Typically, a polypeptide is considered purified when it is at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%) by dry weight free from the naturally occurring polypeptides and molecules with which it is naturally associated. Since a chemically synthesized polypeptide is, by nature, separate from its naturally occurring accompanying components, a synthetic polypeptide is considered purified, but before that it may be stripped of the components used to synthesize the polypeptide (e.g., amino acid residues). With respect to nucleic acid molecules, isolated refers to a nucleic acid molecule that is separated from other nucleic acid molecules that are usually associated with it in the genome.In addition, a nucleic acid molecule. Petition 870260060163, dated 06 / 19 / 2026, page 22 / 128 17 / 53 isolated co may include an engineered nucleic acid molecule such as a recombinant or synthetic nucleic acid molecule.

[0038] Polypeptides can be obtained (e.g., purified) from natural sources (e.g., a biological sample) through known processes such as DEAE ion exchange, gel filtration, and / or hydroxyapatite chromatography. A purified polypeptide can also be obtained, for example, through expression of a nucleic acid molecule in an expression vector or through chemical synthesis. The extent of purity of a polypeptide can be measured using any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. Similarly, nucleic acid molecules can be obtained (e.g., isolated) using routine processes such as, without limitation, recombinant nucleic acid technology (e.g., restriction enzyme digestion and ligation) or the polymerase chain reaction (PCR; see, for example, PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995). In addition, isolated nucleic acid molecules can be chemically synthesized. Virus Particle Manufacturing Processes Exhibiting a Preference for Retrograde Movement in Neurons

[0039] Once a capsid polypeptide has been produced, or once a nucleic acid molecule has been generated and expressed to produce a capsid polypeptide, the polypeptide can be assembled into a virus particle using, for example, a packaging host cell. The components of a virus particle (e.g., rep sequences, cap sequences, inverted terminal repeat (ITR) sequences) can be introduced, transiently or stably, into a packaging host cell using one or more vectors. Although the cap polypeptide Petition 870260060163, dated 06 / 19 / 2026, page 23 / 128 18 / 53 of a virus particle may contain a consensus sequence as described here, the remaining components may be from one or more known AAV serotypes (e.g., AAV2, AAV8, etc.).

[0040] Such virus particles can be purified using routine processes. As used herein, purified virus particles refer to virus particles that are removed from components in the mixture in which they were obtained such as, but not limited to, viral components (e.g., rep sequences, cap sequences), host cell packaging, and partially or incompletely assembled virus particles.

[0041] Once assembled, virus particles can be selected for, for example, the ability to replicate; gene transfer properties; receptor binding ability; and / or seropredominance in a population (e.g., a human population). Determining whether a virus particle can replicate is routine in the technique and typically involves infecting a host cell with a quantity of virus particles and determining whether the virus particles increase in number over time. Determining whether a virus particle is capable of gene transfer is also routine in the technique and typically involves infecting host cells with virus particles containing a transgene (e.g., a detectable transgene such as a reporter gene). Following infection and clearance of the virus, host cells can be evaluated for the presence or absence of the transgene.Determining whether a virus particle binds to its receptor is routine in the technique, and such processes can be performed in vitro or in vivo.

[0042] Determination of seropredominance of a virus particle is routinely performed in the technique and typically includes the use of an immunoassay to determine the predominance of one or more antibodies in samples (e.g., blood samples) from a pair Petition 870260060163, dated 06 / 19 / 2026, page 24 / 128 19 / 53 particular population of individuals. Seropredominance is understood in the art to refer to the proportion of subjects in a population that is seropositive (i.e., has been exposed to a particular pathogen or immunogen), and is calculated as the number of subjects in a population that produces an antibody against a particular pathogen or immunogen divided by the total number of individuals in the population examined. Immune assays are well known in the art and include, without limitation, immunodot, Western blot, enzyme immunoassays (EIA), enzyme-linked sorbent immunoassay (ELISA), or radioimmunoassay (RIA). Similarly, various processes for determining the extent of neutralizing antibodies in a serum sample are available. For example, a neutralizing antibody assay measures the titer at which an experimental sample contains an antibody concentration that neutralizes infection by 50% or more as compared to a control sample without antibody. See also Fisher et al.(1997, Nature Med., 3:306-12); and Manning et al. (1998, Human Gene Ther., 9:477-85). Virus Usage Processes Exhibiting a Preference for Retrograde Movement in Neurons

[0043] A virus or a portion thereof as described herein may be used in a number of research and / or therapeutic applications. For example, a virus or a portion thereof as described herein may be used in human or animal medicine for gene therapy (e.g., in a vector or vector system for gene transfer) or for vaccination (e.g., for gene presentation). More specifically, a virus or a portion thereof as described herein may be used for gene addition, gene augmentation, gene release of a therapeutic polypeptide, gene vaccination, gene silencing, gene editing, gene therapy, RNAi release, cDNA release, mRNA release, miRNA release, sponge formation. Petition 870260060163, dated 06 / 19 / 2026, page 25 / 128 20 / 53 miRNA, genetic immunization, optogenetic gene therapy, transgenesis, DNA vaccination, or DNA immunization.

[0044] A host cell can be transduced or infected with a virus or a portion thereof in vitro (e.g., growing in culture) or in vivo (e.g., in a subject, e.g., a human or a non-human). Host cells that can be transduced or infected with a virus or its portion in vitro are described herein; host cells that can be transduced or infected with a virus or its portion in vivo include, without limitation, projection neurons (e.g., corticopontine projection neurons, sympathetic projection neurons, central nervous system projection neurons), afferents to the dorsomedial striatum (DMS), spinal fungal / cortical neurons, precerebellar neurons, basal ganglia inputs, prethalamic neurons, motor neurons, sensory neurons, or other neurons or cells of the central nervous system.

[0045] A virus or a portion thereof as described herein may be modified to include a payload. A payload typically includes at least one nucleic acid (e.g., a promoter sequence operably linked to a coding sequence that encodes the payload). In certain examples, a payload may be an inhibitory nucleic acid. Inhibitory nucleic acids are known in the art and include, for example, antisense oligonucleotides, short interfering RNAs (siRNAs), and RNA interference molecules (RNAi). In certain examples, a payload may be one or more protein-coding genes. Without limitation, protein-coding genes include an optical reporter construct, a therapeutic gene, or an effector protein.

[0046] Many types of optical reporter constructions are known in the art, and the following list is intended to be representative. Petition 870260060163, dated 06 / 19 / 2026, page 26 / 128 21 / 53 and not exhaustive. For example, optical reporter constructs include, without limitation, GCaMP6 (GCaMP6s, GCaMP6m, or GCaMP6f; see WO 2014 / 059154), a fluorophore (e.g., green fluorescent protein (GFP), enhanced GFP (EGFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), tdTomato), a color-changing construct (e.g., a payload expressing one reporter in one cell population and a different reporter in another population), glucose sensors (e.g., US 2015 / 0111222), iRCaMP (US Patent No. 9,644,007), iRGECO (e.g., US Patent No. 9,644,007), CaMPARI (e.g., US Patent No. 9,518,996), voltage indicators, secondary messengers, receptor signalers, transcription reporters, epigenetic reporters, and neuromodulatory reporters.

[0047] Therapeutic genes are also known in the art and will be dependent on the particular disease or disorder being treated. For example, a therapeutic gene might be for the treatment of a neurodegenerative disorder. Representative therapeutic genes (or the encoded polypeptide) and their associated neurodegenerative disorders include, without limitation, HSP104 for the treatment of Alzheimer's disease, Ataxis (e.g., frataxin) for the treatment of Friedreich's ataxia, lysosomal glucocerebrosidase (GBA) for the treatment of Parkinson's disease, PolyQ-binding protein for the treatment of Huntington's disease, survival motor neuron 1 for the treatment of spinal muscular atrophy, and amyotrophic lateral sclerosis (ALS), autism, dementia, peripheral neuropathy, schizophrenia, and retinal degeneration. In some examples, the payload may be a therapeutic fraction (as opposed to a gene).Therapeutic fractions include, for example, an antibody or its fragment, an immunomodulatory protein, or an RNA interference (RNAi) molecule.

[0048] As used herein, an effector protein refers to which Petition 870260060163, dated 06 / 19 / 2026, page 27 / 128 22 / 53 any type of protein that provides an effect on a cell or its contents (e.g., nucleic acids, proteins, organelles, or processes involving any of the above). For example, effector proteins include, without limitation, recombinases (e.g., Cre or Flp), gene editing systems (e.g., CRISPR / Cas9, TALEN, zinc finger nuclease), optogenetic reagents (activators (e.g., rhodopsin channel or its variants) or inhibitors (e.g., halorhodopsin or Arch)), chemogenetic reagents (e.g., activator / inhibitor versions of the DREADD or PSAM / PSEM systems), activators and / or inhibitors of cell system pathways, and enzymes for epigenetic control.

[0049] It will be appreciated that it may be desirable to release a viral protein that complements or inhibits the function of a virus (e.g., a virus other than AAV). For example, a viral protein that is an entry receptor (e.g., the rabies G protein, a protein that relates to cellular and / or transcellular transport).

[0050] Any number of promoters can be used to drive the sequence encoding the payload. Constitutive promoters are known in the art, as are tissue-specific promoters (e.g., neuron-specific promoters). Simply by example, the promoter could be a synapsin-1, CMV, GFAP, CAG, CaMKII, MBP, EF1alpha, mDIx, or TRE promoter.

[0051] A virus or a portion thereof, usually suspended in a physiologically compatible carrier, may be administered to a subject (e.g., a human or non-human subject (e.g., a primate, a rodent, a reptile, or a bird)). Suitable carriers include saline solution, which may be formulated with a variety of buffer solutions (e.g., phosphate-buffered saline), lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, and water. The virus or its portion is administered in quantities Petition 870260060163, dated 06 / 19 / 2026, page 28 / 128 23 / 53 sufficient factors to transduce or infect cells and to provide sufficient levels of gene transfer and expression to provide a therapeutic benefit without undue adverse effects. Conventional and therapeutically acceptable routes of administration include, but are not limited to, intracranial, intraspinal, or intramuscular injection. Additional routes of administration include, for example, orally, intranasally, intrathecally, via inhalation, intravenously, intraocularly, subcutaneously, intradermally, transmucosally, or via other routes of administration. Routes of administration may be combined if desired.

[0052] The dose of the virus or its portion administered to a subject will depend primarily on factors such as the condition being treated, and the age, weight, and health of the subject. For example, a therapeutically effective dosage of a virus or its portion to be administered to a human subject is generally in the range of about 0.1 mL to about 10 mL of a solution containing concentrations of about 1x10¹ to 1x10¹² genetic copies (GCs) of virus (e.g., about 1x10³ to 1x10⁹ GCs). Transduction and / or expression of a transgene can be monitored at various time points following administration through DNA, RNA, or protein assays. In some examples, transgene expression levels can be monitored to determine the frequency and / or amount of dosing.

[0053] Significantly, the AAV-retro particles described here exhibit up to two orders of magnitude greater retrograde access to corticopontine projection neurons than, for example, canine adenovirus-2 (CAV-2), and retrograde access to certain neurons (e.g., corticopontine projection neurons or afferents to the dorsomedial striatum (DMS)) is comparable to that observed with a synthetic tracer such as Fluoro-Gold fluorescent beads.

[0054] According to the present invention, it is possible to employ Petition 870260060163, dated 06 / 19 / 2026, page 29 / 128 24 / 53 of conventional molecular biology, microbiology, biochemistry, and recombinant DNA techniques within the knowledge of the art. Such techniques are fully explained in the literature. The invention will be further described in the following examples, which do not limit the scope of the processes and compositions of matter described in the claims. Examples Example 1 - Experimental Procedures

[0055] All procedures were performed in accordance with protocols approved by Janelia Research Campus and the University of California Berkeley Institutional Animal Care and Use Committees. Example 2 - Library generation and viral production

[0056] Four previously generated viral libraries were used at the beginning of the targeted evolution procedure: 1) a random mutagenesis library generated by subjecting the AAV2 cap gene (encoding viral proteins VP1-3 and activation assembly protein (AAP)) to error-prone PCR (Maheshri et al., 2006, Nat. Biotechnol., 24:198-204); 2) a library of AAV2 cap gene variants containing 7-mer peptide insertions between N587 and R588 (Müller et al., 2003, Nat. Biotechnol., 21:1040-6); 3) a library of AAV2 cap gene variants containing randomized loop regions (Koerber et al., 2009, Mol. Ther., 17:2088-95); and 4) a DNA shuffling library generated from wild-type AAV1, AAV2, AAV4, AAV5, AAV6, AAV8, and AAV9 cap gene sequences (Koerber et al., 2008, Mol. Ther, 16:1703-9).Each mutant DNA cluster was originally subcloned into the replication-competent AAV packaging plasmid to create a viral plasmid library that, when packaged into AAV virions, can be selected for any new property or function. The replication-competent AAV system incorporates the mutant cap gene into the viral payload, and thus, the genotype of each variant is included. Petition 870260060163, dated 06 / 19 / 2026, page 30 / 128 25 / 53 linked to its phenotype. Capsid sequences of the desired property can then be recovered by DNA sequence analysis of the encapsulated AAV genome.

[0057] The four AAV libraries competent for replication were packaged by transient transfection with calcium phosphate of HEK293 T cells followed by viral harvesting, centrifugation with an iodixanol gradient, and Amicon filtration (Maheshri et al., 2006, Nat. Biotechnol., 24:198-204). Example 3 - In vivo tracer or virus injections

[0058] For localized in vivo viral release, mice or rats were anesthetized with isoflurane (~2% by volume in O2; Surgi Vet, Smiths Medical) and a small hole was drilled in the skull above the required injection site (see Table 1). For some injection sites, multiple injections were made at different depths (see Table 1). For viral injections, 50–100 nL (mouse) or 250–500 nL (rat) of virus-containing solution were slowly injected at each depth into the tissue. For tracer injections, 50 nL of Fluoro-gold 5% (Fluorochrome, Denver, CO) in 0.9% NaCl, or 100 nL of retro-pearls (LumaFluor, Durham, NC) diluted 1:1 in 0.9% NaCl were injected at the same set of sites for each injection target. The injections were made with a pulled glass pipette (broken and beveled to 25-30 µm (outer diameter); Drummond Scientific, Wiretrol II Capillary Microdispenser) counter-filled with mineral oil.An adapted plunger was inserted into the pipette and advanced to displace the contents using a hydraulic manipulator (Narashige, MO-10).

[0059] Piston retraction was used to load the pipette with virus. The injection pipette was positioned with a Sutter MP-285 manipulator. Petition 870260060163, dated 06 / 19 / 2026, p. 31 / 128 26 / 53 Table 1. Coordinates used in this study. Injection coordinates of target injection, mm * SNr mouse ngo A / P: -3.5; M / L: 1.5; D / V: -4.0 and -4.5 mouse cerebellum A / P: 7.1; M / L: 1.4; D / V: -0.8 and -0.5 mouse dorsomedial striatum A / P: 0.5; M / L: 1.5; D / V -2.5 and -2.3 mouse pontine nucleus (BPN) A / P: 0.40; M / L: 0.40; D / V: -5.50, -5.75, and -6.0 mouse dorsomedial striatum ngo A / P 2.16; M / L 1.7; D / V -4.3 rat superior colliculus A / P -6.0; M / L ±1.8; D / V -5.45 All A / P coordinates are given relative to Bregma. Example 4 - Library Selection and Evolution

[0060] The four mutant viral libraries were pooled and injected into either the SNr or cerebellum of adult (6-8 weeks old) wild-type C57 / B16J mice (either sex; Charles River). Three weeks following injections, striatal or metencephalon tissue was similarly removed, DNA was extracted, and virions that successfully reached the remote retrograde target tissue were amplified by PCR and again cloned into rcAAV-packing plasmid to create a new AAV library competent for replication for the next selection cycle. After three selection steps, the rescued cap genes were randomly mutated by error-prone PCR using 5'-ACG CGG AAG CTT CGA TCA ACT ACG CAG-3' (SEQ ID NO:79) and 5'-AGA CCA AAG TTC AAC TGA AAC GAA TTA AAC GG-3' (SEQ ID NO:80) as forward and reverse primers, respectively.

[0061] Two additional in vivo selection cycles were then performed. Individual isolates were sequenced after cycles 4 and 5 to assess the degree of library enrichment (Table 2).

[0062] Convergence of peptide insertion sequences for isolated variants in cycles 4 and 5 Petition 870260060163, dated 06 / 19 / 2026, page 32 / 128 27 / 53 Clone SEQ ID NO of clone (nucleic acid / protein) Loop SEQ ID NO E36G E67A V182 S207G N382D N408S 4R-H-3 3 / 4 LAISDQTKHA 81 4R-H-4 5 / 6 LAISDQTKHA 81 4R-H-5 7 / 8 LAKDQTKSTA 82 X 4R-H-9 9 / 10 LANQDYTKTA 83 4R-H-10 11 / 12 LAISDQTKHA 81 4R-H-12 13 / 14 LANQDYTKTA 83 X 4R-H-15 15 / 16 LAISDQTKHA 81 4R-H-16 17 / 18 LAHDITKNIA 84 X 4R-H-17 19 / 20 LAHDITKNIA 84 4R-SD-12 29 / 30 LAISDQTKHA 81 5R-Hind1 37 / 38 LAQPDATKNA 86 XI 5R-Hind2 39 / 40 LAHDITKNIA 84 XX Petition 870260060163, 19 / 06 / 2026, pág. 33 / 128 28 / 53 Clone SEQ ID NO of clone (nucleic acid / protein) Loop Loop SEQ ID NO E36G E67A V182 S207G N382D N408S 5R-Hind3 41 / 42 LANQDYTKTA 83 X 5R-Hind6 485 / indYT4 LAD 45 / 46 LANQDYTKTA 83 X 5R-Hind10 47 / 48 LADQDYTKTA 85 XX 5R-Hind11 49 / 50 LANQDYTKTA 83 XX 5R-Hind12 51 / 52 LAHDIT / 84R-514R LAISDQTKHA 81 5R-Hind15 55 / 56 LAISDQTKHA 81 5R-Hind16 57 / 58 LAHDITKNIA 84 X 5R-Hind17 59 / 60 LADQDYTKTA 85 XX 5R-61 / Hind1 5R-Hind19 ​​63 / 64 LAHDITKNIA 84 XXX 5R-Hind22 65 / 66 LANQDYTKTA 83 X 5R-Hind23 67 / 68 LAHDITKNIA 84 XXX 5R-Ce1 R LAN 69 / e X5K 71 / 72 LADQDYTKTA 85 5R-Ce6 73 / 74 LAQPDATKNA 86 XX 5R-Ce8 75 / 76 LAKDQTKSTA 82 5R-Ce9 77 / 78 LANQDYTKTA 83 XXX Petition 870260060163, dated 6 / 19 / 2026, p. 34 / 128 29 / 53 -to be continued- Clone T567A Q598L I648V V708I M235L F284Y S492 N496 outro 4R-H-3 X 4R-H-4 X X D L59P 4R-H-5 X 4R-H-9 P 4R-H-10 X Y 4R-H-12 X 4R-H-15 X 4R-H-16 X 4R-H-17 X 4R-SD-2 X 4R-SD-3 X 4R-SD-10 X 4R-SD-11 X P 4R-SD-12 X Q186R,D594N 4R-SD-15 X X X 4R-SD-16 X D69E 4R-SD-17 X Y 4R-SD-18 X 5R-Hind1 X 5R-Hind2 X E12V,K137E Petição 870260060163, de 19 / 06 / 2026, pág. 35 / 128 30 / 53 Clone T567A Q598L I648V V708I M235L F284Y S492 N496 outro 5R-Hind3 X R585G,S662C 5R-Hind6 X 5R-Hind8 X 5R-Hind10 X 5R-Hind11 X N335K 5R-Hind12 X 5R-Hind14 X S423N 5R-Hind15 X N214D 5R-Hind16 X 5R-Hind17 X 5R-Hind18 X 5R-Hind19 X H38L,E147A 5R-Hind22 X P N227D,N587S 5R-Hind23 X 5R-Ce1 X P D41G,K105E,G163D 5R-Ce5 X X 5R-Ce6 X P29A 5R-Ce8 X 5R-Ce9 X P Petição 870260060163, de 19 / 06 / 2026, pág. 36 / 128 31 / 53

[0063] Seventeen variants were selected for secondary selection at the end of cycle 5, and the corresponding cap gene sequences were cloned again into an auxiliary rAAV plasmid. Individual high-titer preparations of the wild-type AAV2 parent and of each of the 17 selected mutant variants carrying a CMV-EGFP payload were then performed by Vector BioLabs, Inc. (Philadelphia, PA). The CMV promoter is typically weak in neurons, and thus, this secondary selection provided a rigorous test of retrograde transport efficiency. The individual AAV variants were injected into both the cerebellum and the globus pallidus. After 3 weeks, endogenous, unamplified EGFP fluorescence was visualized in the regions expected to be labeled if retrograde transport was efficient. Mutant 5R-Hind6 (SEQ ID NO: 43 encoding SEQ ID NO: 44) showed the strongest retrograde transport in both circuits, and was thus chosen for further analysis and nicknamed rAAV2-retro. Example 5 - Heparin Binding Assay

[0064] The heparin affinity of retro- and wild-type AAV2 was analyzed as previously described (Jang et al., 2011, Mol. Ther., 19:667-75). In summary, approximately 1011 purified genomic particles were loaded onto a 1 mL HiTrap heparin column (GE Healthcare Sciences, Piscataway, NJ) previously equilibrated with 150 mM NaCl and 50 mM Tris at pH 7.5. Elution was then performed by increasing the NaCl concentration in 50 mM steps to a final concentration of 950 mM, followed by washing with 1 M NaCl. A small fraction of each elution was used to infect HEK293T cells, and the percentage of GFP-positive cells was quantified 48 hours post-infection using a Guava EasyCyte 6HT flow cytometer (EMD / Millipore). Example 6 - Payloads Used in the Study

[0065] For all subsequent experiments, the promoter Petition 870260060163, dated 06 / 19 / 2026, page 37 / 128 32 / 53 CMV was replaced with a promoter known to be more robust in adult neurons. Cre recombinase and the calcium sensor GCaMP6f were driven by the human Synapsin-1 (hSyn1) promoter. All fluorophores were driven by the CAG promoter, and the color-changing construct was driven by the EF1-alpha promoter. Example 7 - Virus Production for Quantifying Backward Efficiency

[0066] hSyn-Cre payload was packaged using capsids of AAV1, AAV2, AAV5, AAV8, AAV9, DJ, and AAV2-retro were tested in the Janelia Viral Shared Resource. All seven virus preparations were processed in parallel and titered before in vivo injections. All batches were diluted to the lowest measured titer (1.3E12 GC / mL), and each virus was injected into the right pontine nucleus of three adult Rosa26Lox-STOP-LoxH2B-GFP mice (He et al., 2012, Neuron, 73:35-48). Example 8 - Histology

[0067] Animals were sacrificed three weeks following virus injections, at which point the brains were harvested and the right hemisphere was sectioned sagittally to a thickness of 50 micrometers. The sections were mounted on VECTASHIELD Antifade Mounting Medium containing DAPI (Vector Laboratories), and imaged using a PE Pannoramic slide scanner (3D Histech) using a 20x objective and FITC and DAPI filters. Example 9 - Quantification of Retrograde Transport

[0068] Images obtained with the Panoramic slide scanner were stitched together and then analyzed using custom support logic written in Matlab (Mathworks) to detect GFP-labeled nuclei through the cortex. A region of interest (ROI) was manually removed around the cortex to isolate the area in the image for automatic cell counting. To improve nucleus detection, Petition 870260060163, dated 06 / 19 / 2026, page 38 / 128 33 / 53 The image was subjected to convolution with a Mexican Hat kernel comprising the difference of two Gaussians (variance of 26.00 pm and variance of 3.25 pm). Image noise was reduced using an average filter, and basic peak detection was then performed. Example 10 - Retrograde Transport Generality Analyses

[0069] Rosa26-LSL-H2B-GFP mice were injected with nL of rAAV2-retro hSyn1-Cre into the dorsal striatum. Three weeks after injection, coronally sectioned brains were imaged using a Pannoramic Scanner to visualize DAPI-stained nuclei and green fluorescence of H2BGFP-expressing nuclei. The green channel was subjected to convolution with a two Gaussian difference, and peaks were then detected as local maxima on these boundary images using customary functions written in Matlab. The blue channel of each section was aligned to Nissl images from the Allen Brain Institute standardized mouse brain atlas using usual analysis routines written with the aid of Matlab Image Processing Toolbox. Annotated regions from the ABI mouse brain atlas were then used to assign neurons detected in aligned sections to specific brain regions.

[0070] It has been noted that finite precision of the reference atlas along with anatomical variability of individual brains limits the robustness of this semi-automatic process for prominent afferent inputs. Example 11 - In vivo Image Formation of Neuronal Population Activity Following Retrograde Release of GCaMP6f

[0071] Seven adult mice were anesthetized with isoflurane (2%) and placed in a stereotactic frame (Kopf Instruments; Tujunga, CA) on a heating pad at 37°C. The scalp and periosteum over the skull were removed, a layer of UV-curing OptiBond adhesive (Kerr; Orange, CA) was applied, and Petition 870260060163, dated 06 / 19 / 2026, p. 39 / 128 34 / 53 A fabricated headpost – traditional (Osborne and Dudman, 2014, PLoS One, 9(2):e89007) was affixed with dental cement. A retro-AAV2 carrying a hSynGCaMP6f payload was injected into BPN (3.9 mm posterior and 0.4 mm lateral to Bregma, depths of 5.8, 5.6, and 5.4 mm, 100 nL at each depth) using a Nanoliter 2010 injector (WPI). A cranial window (a 170-micrometer-thick laser-cut glass panel, 2 mm in diameter) was placed over the primary motor cortex (centered over 0.7 mm anterior and 1.6 mm to Bregma).

[0072] Following surgery, subcutaneous injections of ketoprofen (5 mg / kg) and buprenorphine (0.1 mg / kg; Henry Schein Animal Health; Melville, NY) were administered. Mice were allowed to recover for 1 week following surgery and then rapidly imaged under a 2-photon microscope to assess virus expression. All animals had cells expressing GCaMP6f visually identified in layer V of M1 one week after injection. Then the animals were habituated to direct fixation on a standard apparatus and trained to retrieve a food pellet as previously described (Guo et al., 2014, Nat. Med., 20:130-8).

[0073] GCaMP6f was excised at 920 nm (typically 20-40 mW at the back aperture) with a Ti:Sapphire laser (Chameleon, Coherent) and imaged through a Nikon 16x, 0.8-NA lens. Light emission passed through a 565 DCXR dichroic filter (Chroma Technology) and an ET525 / 70m-2p filter (Chroma Technology) and was detected by a GaAsP photomultiplier tube (10770PB-40, Hamamatsu). Images (512 x 512 pixels) were acquired at -30 Hz with resonant explorers using ScanImage logic support. Example 12 - CRISPR / Cas9 Genome Editing

[0074] The CMV promoter in pAAV-CMV-SaCas9-empty (Slaymaker Petition 870260060163, dated 06 / 19 / 2026, page 40 / 128 35 / 53 et al., 2016, Science, 351:84-8) was replaced with hSyn1 to generate empty pAAV-hSyn1-SaCas9. Oligonucleotides encoding sgRNA protospacer sequences were ordered in the usual manner, phosphorylated, hybridized, and ligated into the BsaI restriction sites of empty pAAVhSyn1-SaCas9 to generate pAAV-hSyn1-SaCas9-tdTomato-1 to 10. Oligonucleotide sequences used were: tdTomato sgRNA 1 Fwd: CAC CGC AAG GGC GAG GAG GTC ATC A (SEQ ID NO:108) tdTomato sgRNA 1 Rev: AAA CTG ATG ACC TCC TCG CCC TTG C (SEQ ID NO:87) tdTomato sgRNA 2 Fwd: CAC CGT GGA GGG CTC CAT GAA CGG CC (SEQ ID NO:88) tdTomato sgRNA 2 Rev: AAA CGG CCG TTC ATG GAG CCC TCC AC (SEQ ID NO:89) tdTomato sgRNA 3 Fwd: CAC CGA GGA CGG CGG CCA CTA CCT GG (SEQ ID NO:90) tdTomato sgRNA 3 Rev: AAA CCC AGG TAG TGG CCG CCG TCC TC (SEQ ID NO:91) tdTomato sgRNA 4 Fwd: CAC CGA CAA CAA CAT GGC CGT CAT CA (SEQ ID NO:92) tdTomato sgRNA 4 Rev: AAA CTG ATG ACG GCC ATG TTG TTG TC (SEQ ID NO:93) tdTomato sgRNA 5 Fwd: CAC CGA AGG ACG GCG GCC ACT ACC TGG (SEQ ID NO:94) tdTomato sgRNA 5 Rev: AAA CCC AGG TAG TGG CCG CCG TCC TTC (SEQ ID NO:95) tdTomato sgRNA 6 Fwd: CAC CGA CAA CAA CAT GGC CGT CAT CA (SEQ ID NO:96) tdTomato sgRNA6 Rev: AAA CTG ATG ACG GCC ATG TTG TTG TC (SEQ ID NO:97) Petition 870260060163, 06 / 19 / 2026, p. 41 / 1 36 / 53 tdTomato sgRNA 7 Fwd: CAC CGG TCA CCT TCA GCT TGG CGG T (SEQ ID NO:98) tdTomato sgRNA 7 Rev: AAA CAC CGC CAA GCT GAA GGT GAC C (SEQ ID NO:99) tdTomato sgRNA 8 Fwd: CAC CGC CGT ACA TGA ACT GGG GGG A (SEQ ID NO:100) tdTomato sgRNA 8 Rev: AAA CTC CCC CCA GTT CAT GTA CGG (SEQ ID NO:101) tdTomato sgRNA 9 Fwd: CAC CGT CTT GTA ATC GGG GAT GTC GG (SEQ ID NO:102) tdTomato sgRNA 9 Rev: AAA CCC GAC ATC CCC GAT TAC AAG AC (SEQ ID NO:103) tdTomato sgRNA 10 Fwd: CAC CGC CGT CCT GCA GGG AGG AGT C (SEQ ID NO:104) tdTomato sgRNA 10 Rev: AAA CGA CTC CTC CCT GCA GGA CGG C (SEQ ID NO:105) .

[0075] The ability of each oligo to target genome editing was first evaluated in vitro. Neuro2A cells were transfected with 800 ng of pAAV-hSyn1-SaCas9-tdTomato-1 to -10, 100 ng of pAAV-FLEX-CAGtdTomato, and 100 ng of pAAV-CAG-EGFP using polyethyleneimine. 72 hours after transfection, cells were harvested and ~70,000 EGFP-positive Neuro2A cells were isolated by fluorescence-activated cell sorting (FACS) using a BD Influx Sorter (BD Biosciences). Genomic DNA was then extracted, and the frequency of tdTomato gene modification was assessed using the Surveyor nuclease assay (Integrated DNA Technologies) as previously described (Cong et al., 2013, Science, 339:819-23). sgRNA7 - one of two that appeared to direct two cleavage events within the tdTomato sequence - was packaged into retro-AAV2 and used for in vivo genome editing. Petition 870260060163, dated 06 / 19 / 2026, page 42 / 128 37 / 53

[0076] ~100 nL (5x1013 vector genomes (vg) / mL) of AAV2retro-hSyn1-SaCas9-tdTomato or AAV2retro-hSyn1-SaCas9-empty were then injected into the BPN of Rbp4-Cre x tdTomato mice as described above. Six weeks after injections, brains were harvested and 40 µm thick coronal sections were cut and stained against the HA-labeled Cas9 (Cell Signaling anti-HA C29F4 antibody, diluted 1:1600; secondary antibody: Alexa Fluor 488 anti-dumb mouse (1:250; Jackson ImmunoResearch)) and against the neuronal marker NeuN (Millipore anti-NeuN A60 antibody, diluted 1:250; secondary antibody: Alexa Fluor 647 anti-dumb rabbit (1:500; ThermoFisher, A-31573)). Following antibody staining, sections were mounted on slides with VECTASHIELD Antifade Mounting Medium containing DAPI (Vetor Laboratories) and visualized using a Zeiss Axio Observer A1 inverted microscope (Zeiss). Quantification of immunostaining was performed using ImageJ analysis logic support (NIH). Example 13 - Targeted evolution of rAAV2-retro

[0077] To engineer new rAAVs with improved retrograde transport, an in vivo targeted evolution approach was designed that enriched for rAAV capsid variants that were efficiently transported to neuronal cell bodies by sending long-band projections to the virus injection site in the mouse brain (Figure 1A). To maximize the probability of recovering a variant with the desired properties, a diverse mixture of previously described libraries of rAAV capsid variants (Koerber et al., 2008, Mol. Ther., 16:1703-9; Koerber et al., 2009, Mol. Ther., 17:2088-95; Koerber et al., 2006, Nat. Protocols, 1:701-6; Müller et al., 2003, Nat. Biotechnol., 21:1040-6) was used as starting material. Viral particles were packaged in a way that linked each variant capsid to the AAV genome containing the Petition 870260060163, dated 06 / 19 / 2026, p. 43 / 128 38 / 53 corresponding cap gene, and the final assembly of capsid variants included point mutants, variants with a random 7-mer peptide insertion in the AAV2 region used to bind their co-receptor, heparan sulfate, and random chimeras of capsid gene sequences from seven related serotypes (Figure 1A). To identify variants with broad retrograde tropism, two independent populations of projection neurons were targeted: striatal GABAergic neurons projecting to the substantia nigra pars reticulata (SNr) and metencephalon glutamatergic neurons projecting to the cerebellar cortex. Three weeks following injection of the entire assembly of rAAV variants into the SNr or cerebellum (one injection per animal), striatal tissue or metencephalon, respectively, it was removed, the cap sequences were recovered by PCR, and the virus was repackaged (Figure 1B).After two more selection stages, error-prone PCR was performed to further diversify the library, followed by two final in vivo selection stages.

[0078] 30 cap variants were sequenced after the 4th evolutionary cycle, and most originated from the insertion library and contained exogenous 7-mer peptides between N587 and R588 of the wild-type AAV2 VP1 capsid gene. Intriguingly, all recovered insertions were of the form LAxxDxTKxA (SEQ ID NO: 106) or LAxDxTKxxA (SEQ ID NO: 107) (where the italicized, bold residues are from the insertion); mutations at other sequence locations were also enriched (Table 2). An additional 22 clones were sequenced after the 5th evolutionary cycle, and with this additional evolutionary cycle, all sequences were AAV2 mutants with LAxxDxTKxA (SEQ ID NO: 106) / LAxDxTKxxA (SEQ ID NO: 107) insertions (Table 2), demonstrating a marked degree of convergence. This convergence suggested that specific peptide insertions in the heparin-binding loop were largely responsible for the functionality. Petition 870260060163, dated 06 / 19 / 2026, p. 44 / 128 39 / 53 retrograde age, with potential secondary contributions from other sites.

[0079] A secondary selection of seventeen isolated variants possessing different combinations of peptide insertions and point mutations in the capsid sequence was examined below. To apply a high level of rigor, the selected variants were packaged with an enhanced green fluorescent protein (EGFP) transgene directed by the CMV promoter, which is typically weak in neurons. For each capsid variant, its ability to release sufficient payload to cell bodies in key afferent regions to allow detection of a non-antibody amplified EGFP signal three weeks after injection was evaluated. The clone (LA DQDYTKTA insertion (SEQ ID NO:85) + V708I + N382D) that showed the strongest retrograde transport in two independent circuits in this secondary selection (cortex to Globus Pallidus and inferior olive / basal pontine nucleus to cerebellum) was chosen for further analysis and dubbed rAAV2-retro.When two additional promoters more commonly used in in vivo rodent studies were evaluated (CAG - Figure 1B, or human Synapsin-1, data not shown), remarkable retrograde labeling efficiency was observed with this rAAV variant in a range of different circuits in mice and rats (Figure 1B and Figure 7). Insertion swapping of 7-mer to one of the other recovered sequences, or addition of additional point mutations identified in the selection, does not lead to further increases in retrograde transport. Example 14 - Efficient Retrograde Access for Projection Neurons

[0080] Within descending motor pathways, the cortical tract The pontine pathway is remarkably convergent and is known to contribute over 95% of afferents to the basal pontine nuclei (BPN). This pathway thus represents a particularly advantageous system for quantifying viral uptake efficiency and retrograde transport. Petition 870260060163, dated 06 / 19 / 2026, page 45 / 128 40 / 53 by axonal terminals of projection neurons. Indeed, retro-rAAV2 injection into BPN resulted in dense labeling of layer V neurons (Figure 2A), consistent with past research studies (Legg et al., 1989, J. Comp. Neurol., 286(4):427-41).

[0081] Next, the efficiency of retrograde transport in the corticopontine circuit for rAAV2-retro vs. several commonly used AAV serotypes was compared under identical infection and processing conditions (Figures 2B-D). To ensure quantification accuracy, and to eliminate possible confounding from cell-to-cell variability at the transgene expression level, AAV was used to release Cre recombinase in Rosa26-LoxSTOP-Lox-H2B-GFP transgenic mice (He et al., 2012, Neuron, 73:35-48). It was shown that even a low concentration of the Cre enzyme is sufficient to trigger the expression of such a Cre-dependent cassette, and the tight nuclear localization of the fused-histone reporter provides unequivocal identification of infected cells, without confounding neuropil signal.

[0082] A semi-automated analysis procedure was used to calculate the linear density of infected cortical projection neurons in sagittal sections imaged from mouse brains harvested three weeks after local virus injection into BPN (Figure 2C). For animals infected with rAAV2, minimal cortical GFP expression was observed (linear density 0.98 + / - 0.20 neurons / mm, mean + / - sem, n = 5; Figure 2B, middle panel). In contrast, and in agreement with the previous observation (Figure 2A), a dense layer of GFP-positive layer V projection neurons could be observed throughout the rostrocaudal axis of the cortex in animals injected with rAAV2-retro (linear density 130.11 + / - 11.08 neurons / mm, n = 4; Figure 2B, bottom panel). None of the other commonly used AAV serotypes, nor canine adenovirus-2, matched the retrograde efficiency of the retro-engineered rAAV2 variant (linear density). Petition 870260060163, dated 06 / 19 / 2026, page 46 / 128 41 / 53 AAV1: 0.05 ± 0.04, AAV2: 0.98 ± 0.2, AAV5: 2.38 ± 1.24, AAV8: 1.43 ± 1.43, AAV9: 1.98 ± 0.86, DJ: 24.82 ± 14.32, CAV-2: 5.56 ± 4.13, n=3 to 5 each; Figure 2D, Figure 8). Furthermore, the density of cortical projection neurons labeled by rAAV2-retro was comparable to that obtained with Fluoro-Gold (Schmued and Fallon, 1986, Brain Res., 377:14754), a robust synthetic retrograde tracer (linear density 81.03 + / - 11.08 neurons / mm, n=3). Thus, rAAV2-retro exhibits two-orders-of-magnitude improvement over existing serotypes in retrograde access to corticopontine projection neurons, and rivals the efficacy of synthetic retrograde tracers. Example 15 - Generality of Retrograde Functionality

[0083] Whether the retrograde functionality of rAAV-retro extended to other circuits was examined next, specifically through extension characterization in which it tagged various afferents to the dorsomedial striatum (DMS) – a part of the basal ganglia that receives long-band inputs – from a variety of cortical and subcortical areas. It was found that the effectiveness of retrograde access mediated by rAAV2-retro to the strongest afferent inputs in DMS – cortex, thalamus, and amygdala – was comparable to that of fluorescent beads classically used for retrograde investigation (Figure 3A).To provide an unbiased estimate for the number of retrogradely labeled neurons in all known brain areas to provide significant long-range input to the DMS, an algorithm was developed that determined any fluorescent marker detected in a section imaged from a mouse brain to specific brain areas through section alignment to annotated Allen Brain Atlas (Figure 3B-C). Quantitative analyses (Figure 3C) revealed that strong retrograde labeling was verified in a vast majority of regions that were previously reported to send prominent projections to. Petition 870260060163, dated 06 / 19 / 2026, page 47 / 128 42 / 53 the striatum (Pan et al., 2010, Front Neuroanat., 4:147). In a notable exception, only modest staining was observed in the substantia nigra pars compacta, despite it being the source of a strong dopaminergic input to DMS (Figure 3C, arrow pointing to cell count for SNc). A small subset of projection neuron classes was found to be similarly refractory to retrograde access by rAAV2-retro in some of the other circuits tested (Table 3; note that all other AAV serotypes tested also failed to stain these projections). Despite these exceptions, rAAV2-retro is widely applicable within the central nervous system. Table 3. Efficiency of rAAV2-retro in various circuits. Low efficiency, rather than the absence of retrograde transport in corticothalamic and corticocollicular projections, is suggested by efficient labeling of projection neurons following local release of Cre-dependent payloads to projection neuron cell bodies. Petition 870260060163, dated 06 / 19 / 2026, page 48 / 128 43 / 53 Injection site | Targeted projection neuron location | Labeling efficiency | Mouse mPFC (IL / PL) transgene | ECM | Strong | Cre | mPFC (IL / PL) mouse | ECM | Strong | Cre | mPFC (IL / PL) mouse | Contra-mPFC | Strong | Cre | mPFC (IL / PL) mouse | Dorsal mid-thalamus | Moderate | Cre | mPFC (IL / PL) mouse | Thalamic nucleus reuniens | Moderate | Cre | mPFC (IL / PL) mouse | Insular cortex | Strong | Cre | mPFC (IL / PL) mouse | Hippocampus - Ventral CA1 | Strong | Cre | mPFC (IL / PL) mouse | AcG | Strong | Cre | OFC ECM | Strong | Cre | OFC ECM | Strong | Cre | OFC contra-OFC | Strong | Cre | OFC insular cortex | Strong | Cre | OFC Hippocampus - Ventral CA1 | Moderate | Cre | Dorsal CA1 ECM | Strong | Cre | Dorsal CA2 ECM | Strong | Cre | Dorsal CA3 CA3 | Weak | Cre | Dorsal CA4 thalamic nucleus reuniens | Weak | Cre | Dorsal CA4 thalamic nucleus reuniens | Weak | Cre | Amygdala mPFC Strong Cre Lateral hypothalamus mPFC Strong Cre Dorsomedial striatum mPFC Strong Cre Petition 870260060163, dated 06 / 19 / 2026, page 49 / 128 44 / 53 Injection site Targeted projection neuron location Labeling efficiency Ventral striatum transgene (NAcc) mPFC strong Cre MEC mPFC Moderate FLPo mPFC CA1 ventral moderate FLPo mPFC MEC Moderate FLPo subiculum nucleus accumbens Moderate Cre subiculum Ventral hippocampus strong Cre subiculum Interantethalamic nucleus Moderate Cre subiculum Retrosplenial cortex strong Cre subiculum Lateral entorhinal cortex Strong Cre subiculum Medial entorhinal cortex strong Cre Superior colliculus layer 5 in visual cortex Very weak Gcamp6s Superior colliculus layer 5 in visual cortex Strong Cre dLGN layer 6 in visual cortex Very weak Gcamp6s dLGN Layer 6 in visual cortex Strong Cre Visual cortex Contralateral visual cortex strong Cre Visual cortex Contralateral visual cortex Strong GFP Visual cortex Contralateral visual cortex weak Gcamp6s Visual cortex Contralateral visual cortex Strong tdtomato Visual cortex LGN strong tdtomato Visual cortex Claustrum Strong tdtomato Visual cortexCingulate moderate tdtomato Petition 870260060163, dated 06 / 19 / 2026, p. 50 / 128 45 / 53 Injection site Targeted projection neuron location Labeling efficiency Transgene Visual cortex LM Strong tdtomato Dorsolateral striatum M1 strong tdtomato Dorsolateral striatum M1 moderate GFP Dorsolateral striatum M2 strong tdtomato Dorsolateral striatum Somatosensory cortex Strong tdtomato Dorsolateral striatum Basolateral amygdala strong tdtomato Dorsolateral striatum Insular cortex Strong tdtomato Dorsolateral striatum Entorhinal cortex strong tdtomato Dorsolateral striatum Globus Pallidus moderate tdtomato Dorsolateral striatum Thalamus moderate tdtomato Dorsolateral striatum Intralaminar thalamic nucleus Strong GFP Dorsolateral striatum Very weak tdtomato Dorsolateral striatum VTA weak tdtomato Basolateral amygdala Taste cortex Strong flex-tdtomato motor forelimb Thalamus motor Weak GFP nucleus tracts solitary Lateral amygdala moderate tdtomato Spinal cord Cortex Strong flex-tdtomato Spinal cord Cortex strong GFP CordSpinal cord, lamina IX: strong presynaptic inhibitory neurons, moderate muscle motor neurons, proprioceptive neurons. Petition 870260060163, dated 06 / 19 / 2026, page 51 / 128 46 / 53 Example 16 - Retrograde Access to Genetically Defined Neuronal Populations

[0084] It was also determined whether the retrograde functionality of rAAV2-retro can be combined with the specificity of Cre transgenic lines to allow interrogation of specific classes of projection neurons. Specifically, experiments were performed to determine whether rAAV2-retro and Cre transgenic lines can be combined to segregate two functionally distinct band-long connections that run in parallel between two brain areas. Projections from the cerebral cortex to the striatum arise primarily from neurons in layer V, but some neurons in layers II and III also provide striatal inputs. It has been suggested that inputs to the striatum from different cortical layers constitute separate pathways, with neurons in layer V projecting to the striatal plate compartment, and those in layers II and III to the matrix.The interdigitated nature of micro-compartments and plaque makes it difficult to selectively target these pathways for functional interrogation with the rAAV2-retro system alone. Therefore, it was explored whether the two inputs could be segregated by combining rAAV2-retro with a transgenic line that expresses Cre recombinase in all layer V neurons (Gerfen et al., 2013, Neuron, 80:1368-83), but not in neurons in layers II and III (Figure 4A).

[0085] To highlight both pathways in the same experiment, a Cre-dependent color-switching payload was chosen, which expresses tdTomato in the absence of Cre, but reverses to drive EGFP expression in Cre-positive cells (Saunders et al., 2012, Front Neural Circuits, 6:47). When rAAV2-retro carrying this payload was injected into the dorsomedial striatum of a specific Cre-triggering line—layer V, Rbp4_KL100 Cre (Gerfen et al., 2013, Neuron, 80:1368-83), only those layer V neurons showed Petition 870260060163, dated 06 / 19 / 2026, page 52 / 128 47 / 53 projecting to the striatum expressed EGFP. Furthermore, the layer II and III corticostriatal pathway was clearly distinguishable through tdTomato expression (Figure 4B). Consistent with the topographic nature of the corticostriatal projection, highly localized injections of the virus into the dorsomedial striatum led to the labeling of correspondingly small sections of the cortex. The layer V and layer II and III corticostriatal populations, however, were always comarcadal (Figure 4B, lower panel), suggesting that the two pathways from the same cortical area traverse through neighboring matrix and plaque microcompartments within the striatum.

[0086] Although this experiment highlighted both paths, choosing a Cre-on or Cre-off payload will allow selective access to one to the exclusion of the other. This example, therefore, underscores the added circuit interrogation specificity that can be achieved by combining a highly efficient retrograde virus with available Cre (or Flp) trigger lines. Example 17 - Use of rAAV2-retro for Circuit Interrogation and Gene Manipulation

[0087] The usefulness of rAAV2-retro for circuit interrogation will depend on its ability to mediate high levels of expression of genetically encoded cues and effectors. The ability to monitor neural activity in defined classes of projection neurons was first evaluated through rAAV2-retro mediated expression of GCaMP6f (Chen et al., 2013, Nature, 499:295-300) (Figure 5aC). Using two-photon Ca2+ imaging, somatic and dendritic Ca2+ transients were detected in the primary motor cortex as early as 7 days after viral release to BPN (Figure 5D). The temporal profile of Ca2+ signals reflected the structure of a reaching task performed, with the signal in many cortico-point neurons being tightly linked to the cue follow (Figure 5E-F) (Li et al., Petition 870260060163, dated 06 / 19 / 2026, page 53 / 128 48 / 53 (2015, Nature, 519:51-6). Repeated recordings of identified neurons were possible through experiments within an initial session during the expression time course (Figure 5E, F), and through many behavioral sessions over two months post-infection. Thus, rAAV2-rretro confers the ability to express sensors in projection neurons at levels sufficient for imaging, creating many new opportunities to decipher the contributions of specific projections to circuit computations.

[0088] Finally, the utility of rAAV2-retro was evaluated for the delivery of effectors, such as the CRISPR / Cas9 gene editing system, to projection neurons (Figure 6). Specifically, Staphylococcus aureus Cas9 (SaCas9 (Slaymaker et al., 2016, Science, 351:84-8)) and a simple guide RNA designed to remove tdTomato expression were packaged in rAAV2-retro. Delivery of rAAV2-retro-SaCas9-antitdTomato to BPN from animals expressing tdTomato in cortical layer V excitatory neurons resulted in suppression of tdTomato expression in 88.6 + / - 0.7% of layer V neurons expressing SaCas9 (Figure 6B, bottom panel and Figure 6C, n = 3). In contrast, release of untargeted SaCas9 does not lead to any discernible changes, with only 4.4 + / - 3.2% of cells showing potential reduction in tdTomato expression (Figure 6B, top panel, and Figure 6C, n = 3).Furthermore, dtTomato expression remained unchanged in layer V neurons that were inaccessible via pontine injection. The rAAV2-retro system thus allows efficient gene modification selectively in neurons projecting to specific areas of interest.

[0089] Collectively, these observations establish rAAV2retro as an effective reagent for genetically accessing projection neurons for functional interrogation of neuronal circuits and, eventually, for potential therapeutics. Petition 870260060163, dated 06 / 19 / 2026, page 54 / 128 49 / 53 Example 18 - Discussion

[0090] Recombinant adeno-associated viruses can greatly facilitate the functional dissection of mammalian neural circuits and hold promise for therapeutic intervention in nervous system disorders. Targeted evolution has been used to endow the AAV capsid with the additional capacity for efficient retrograde access to projection neurons in many circuits. The newly engineered rAAV-retro offers up to two orders of magnitude improvement in retrograde transport compared to commonly used AAV serotypes, matching the efficacy of synthetic retrograde tracers in many circuits. The level of transgene expression obtained with rAAV2-retro via retrograde access is broad enough for interrogation of neural circuit function, as well as for targeted manipulations of the neuronal genome.Thus, by enabling selective monitoring and manipulation of projection neurons connecting different brain areas, rAAV2-retro-based tools are stabilized to provide insights into how large-scale networks enable brain function, and hold promise for therapeutic intervention in diseases characterized by progressive large-scale network dysfunction.

[0091] The markedly increased retrograde access efficacy provided by rAAV2-retro compared to its parent serotype AAV2 may have been provided through an insertion-mediated disruption of the native heparan sulfate binding site and / or through the creation of a new binding surface that incorporates the inserted peptide. This variant has reduced heparin affinity (Figure 9), which may decrease virus sequestration in the synaptic cleft extracellular matrix and enhance local vector dissemination, as observed with AAV1 and AAV6. However, the resulting increase in vector dissemination alone cannot explain the transport efficacy. Petition 870260060163, dated 06 / 19 / 2026, page 55 / 128 50 / 53 retrograde, insofar as other similarly inserted 7-mer sequences disrupt heparin binding but do not affect retrograde transport. Furthermore, AAV5 and AAV9 do not bind heparin, yet their retrograde transport efficiency is similar to that of AAV2. In support of the alternative explanation, the peptide insertions selected in the original selection (LAxxDxTKxA (SEQ ID NO:106) / LAxDxTKxxA (SEQ ID NO:107)) share the same overall composition, differing simply in the registration of the conserved motif. The engineered peptide insertion may support enhanced binding to an existing cellular cofactor in the AAV pathway (e.g., the recently identified common AAV receptor), or it may create a novel interaction with cellular machinery – a cell surface receptor and / or a component of vesicular trafficking or nuclear entry pathway.

[0092] Despite multiple orders of magnitude improvements in retrograde transport that rAAV2-retro offers over existing serotypes, a small set of projection neuron classes appears refractory to efficient retrograde infection via this newly evolved rAAV variant (Table 3). It should be noted, however, that other AAV serotypes also failed to tag these projections. Whether the expression level of the critical cellular factor that interfaces with this new AAV variant in these neurons is just extremely low—a conclusion supported for the corticothalamic and corticocollicular projections, for example, by observing that rAAV2-retro can still release sufficient Cre recombinase to cell bodies to drive high expression levels for locally released Cre-dependent payloads (Table 3, highlighted entries)—or whether the factor is entirely lost remains to be determined in each case.Further future capsid modifications for this or other variants may allow for improved retrograde transduction of these classes of neurons. Petition 870260060163, dated 06 / 19 / 2026, page 56 / 128 51 / 53

[0093] The rAAV2-retro vector system provides an important addition to the genetic toolkit for dissecting neural circuit function, as gaining access to individual classes of projection neurons will be a critical enabling step in elucidating how local circuit dynamics and large-scale network function are coordinated. Local circuit computations are increasingly thought to depend on the dynamics of the entire neuronal population within a particular local circuit module. How these dynamics map onto the different classes of projection neurons—and thus what information is passed to the different downstream targets—remains unresolved for most circuits. rAAV2-retro based vectors, alone or in combination with specific Cre transgenic lines, allow genetic access to specific populations of projection neurons.In turn, rAAV2-retro-carrying rabies G glycoprotein can be used for trans-complement of a newly developed non-toxic conditional rabies vector for accessing presynaptic microcircuits by impinging on particular classes of projection neurons. The resulting ability to selectively monitor and manipulate the activity of individual projection neuron classes and their local microcircuits should provide insight into how projection neurons translate local circuit dynamics into their respective large-scale networks.

[0094] rAAV2-retro also holds promise for therapeutic intervention, with several possible applications. For example, in situations where pathology impacts large volumes of neural tissue – such as lysosomal storage diseases or Alzheimer's – multiple injections pose a safety risk and may be insufficient to achieve the necessary transduction levels. However, a small number of injections at strategic locations may allow vector dispersion over large volumes (e.g., cortico-ventricular tract – pons). Petition 870260060163, dated 06 / 19 / 2026, page 57 / 128 52 / 53 tino from the point of convergence in the BPN), or difficult-to-access tissue (e.g., spinal motor neurons of muscle). Furthermore, large-scale functional networks have been implicated in the spread of many neurodegenerative disorders from their spatially localized onset. A prominent emerging view posits that deposition of abnormal protein assemblies in vulnerable neuronal populations triggers a pathological cascade of aberrant neuronal activity within, and disintegration of, large-scale functional networks, ultimately leading to failure of neurological functions. Intriguingly, the affected neural networks appear capable of transiently overcoming the aberrant dynamics initially in the disease, whereas patients with many neurodegenerative diseases frequently show periods of dramatic improvement.Thus, initial intervention aimed at reducing the spread of aggregates from the cortical origin of pathology may be sufficient to stabilize, or even restore, cognitive function. From this perspective, neurons projecting subcortically to mesocortical regions where pathological protein aggregates first appear, in both Alzheimer's and Parkinson's disorders, constitute an attractive target for intervention. Access to these projection neurons with retro-rAAV2-based tools designed, for example, to introduce a mutation capable of further preventing aggregation in cis or to release chaperones capable of disassembling the aggregates has the potential to slow the progression of the most debilitating cognitive symptoms. Evaluation of the long-term efficiency and safety of retro-rAAV2 reagents in non-human primates will pave the way for their eventual consideration for these, and other, gene therapy approaches.

[0095] It should be understood that, although the processes and compositions of matter have been described here in conjunction with a Petition 870260060163, dated 06 / 19 / 2026, p. 58 / 128 53 / 53 number of different aspects, the previous description of the various aspects is intended to illustrate and not limit the scope of the processes and compositions of matter. Other aspects, advantages, and modifications are within the scope of the following claims.

[0096] Processes and compositions are shown that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the processes and compositions shown. These and other materials are shown here, and it is understood that combinations, subsets, interactions, groups, etc., of these processes and compositions are shown. That is, although specific reference to each of various individual and collective combinations and permutations of these compositions and processes may not be explicitly shown, each is specifically contemplated and described here. For example, if a particular composition of matter or a particular process is shown and discussed and a number of compositions or processes are discussed, each and every combination and permutation of the compositions and processes is specifically contemplated unless specifically indicated otherwise.Similarly, any subset or combination of these is also specifically considered and shown. Petition 870260060163, dated 06 / 19 / 2026, p. 59 / 128

Claims

1 / 2 CLAIMS 1. Viral capsid protein, characterized in that it comprises a sequence selected from the group consisting of xxDxTKx (SEQ ID NO:1) and xDxTKxx (SEQ ID NO:2), wherein the viral capsid protein consists of an amino acid sequence selected from the group consisting of the SEQ ID NOs: 6, 8, 10, 12, 14, 18, 22, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 54, 56, 60, 64, 66, 68, 70, 72, 74, 76 and 78.

2. Viral particle, characterized in that it comprises the viral capsid protein, as defined in claim 1.

3. Viral particle, according to claim 2, characterized in that retrograde access to corticopontine projection neurons or afferents to the dorsomedial striatum (DMS) is comparable to the synthetic tracer, Fluoro-Gold fluorescent beads.

4. Use of adeno-associated viruses (AAVs) comprising a viral capsid protein, as defined in any one of claims 1 to 3, and payload packaged therein, characterized in that it is used in the manufacture of a medicament or composition for the treatment of a neurodegenerative disorder in an individual.

5. Use according to claim 4, characterized in that the individual is a human.

6. Use according to claim 4, characterized in that the individual is a non-human individual, preferably wherein the non-human individual is selected from among a primate, a rodent, a reptile, and a bird.

7. Viral capsid protein, according to claim 1, characterized in that it consists of an amino acid sequence of SEQ ID NO:

44. Petition 870260060163, dated 06 / 19 / 2026, page 60 / 128 2 / 2 8. Use, according to claim 4, characterized in that the viral capsid protein consists of an amino acid sequence of SEQ ID NO:

44. Petition 870260060163, dated 06 / 19 / 2026, p. 61 / 128

Images