Gene therapy method for controlling organ function

Abstract

Methods and compositions for controlling the function of visceral organs are provided. For example, the methods and compositions can be used for preventing, suppressing, or treating diseases by controlling, for example, regulating organ function. In one embodiment, a viral mediator is delivered to an organ, and the virus infects nerves that regulate organ function. In one embodiment, the viral mediator is a retrograde mediator. In one embodiment, the viral mediator encodes a gene product, and the activity of the gene product is controlled by an exogenously delivered agent or energy. Thus, the delivery of the agent or energy controls organ function.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of the filing date of U.S. Application No. 62 / 712,669, filed Jul. 31, 2018, the disclosure of which is incorporated herein by reference. Background of the Invention

[0003] Gene therapy holds great promise for a variety of neurological diseases. It has recently been recognized that neuronal control of organ function represents an opportunity for therapeutic modulation of organ function by manipulating neuronal activity. Mechanical means for altering neuronal function are currently available, including stimulators that can electrically stimulate the trunk or branches of a nerve, such as the vagus nerve. However, these non-specifically affect all neurons within the stimulated nerve, and they involve complex implants that have the complexities inherent in mechanical devices, including lead migration and infection, while the pulse generator must also be periodically recharged and / or replaced to maintain function.

[0004] Gene therapy agents are capable of targeting neurons to visceral organs, however, injecting viral vectors into ganglia, the brain, or spinal cord regions that house the cell bodies of these neurons will not permit control of a single organ, as these are mostly mixed populations of neurons that send projections to many organs. For example, sensory neurons of the vagus nerve from the stomach can sense stretch and satiety, while sensory neurons from the vagus nerve to the lungs are responsible for the cough reflex. Thus, injecting a gene therapy agent that modulates neuronal function into the nodose ganglion will target both neuronal populations, thereby affecting the function of both organs, which is undesirable when treating cough or metabolic disorders individually.

[0005] Accordingly, there is a need for a means to genetically regulate the function of subsets of neurons in specific organs in order to modulate organ function to improve disease. Summary of the Invention

[0006] The present disclosure provides materials and methods for modulating organ function to prevent, inhibit, or treat diseases. In one aspect, the method provides delivering a viral vector to an organ, and then the viral vector is taken up by axons that regulate the function of those organs. In one embodiment, the viral vector is an adeno-associated virus (AAV) vector. In one embodiment, a retrograde form of adeno-associated virus (AAV) is specifically taken up into a subset of vagal sensory neurons that respond to gastric distension and cause satiety when injected into the gastric wall. Molecules that provide the retrograde vector are known in the art and include, but are not limited to, native viral proteins such as HSV proteins, rabies virus G, glycoprotein C type, VSV G, B19G, pseudorabies virus proteins, AAV capsid proteins, and dynein. This represents a specific subset of neurons that project from the nodose ganglion without affecting other nodose neurons that provide sensation to other visceral organs. The viral vector can also be other forms of retrograde vectors, including, but not limited to, retrograde lentivirus (LV) vectors, herpes simplex virus (HSV) vectors, or canine adenovirus (CAV) vectors.

[0007] In one embodiment, a method is provided for delivering one or more genes to nerve fibers that regulate the function of an organ such as a visceral organ, including, but not limited to, the stomach, small intestine, large intestine, pancreas, liver, spleen, gallbladder, lung, kidney, and heart. In one embodiment, a viral gene therapy vector is delivered to an organ region innervated by a regulatory nerve (such as the vagus nerve, cardiopulmonary nerve, thoracic visceral nerve, lumbar visceral nerve, sacral visceral nerve, or pelvic visceral nerve). In one embodiment, the organ is the stomach, intestine, pancreas, liver, lung, heart, adrenal gland, kidney, gonad, bladder, anal sphincter, or urethral sphincter. In one embodiment, the viral vector is modified for retrograde transport in the central nervous system. In one embodiment, the viral vector is injected into the organ. In one embodiment, delivering the vector prevents, inhibits, or treats a disease. In one embodiment, the viral vector is injected into the stomach, and the expression of the gene controls food intake. In one embodiment, the viral vector is delivered to the lung, for example, by inhalation, and the expression of the gene controls coughing. In one embodiment, the expression of the gene activates a regulatory nerve. In one embodiment, the expression of the gene inhibits a regulatory nerve.

[0008] The present disclosure also provides a viral vector having improved properties for retrograde uptake into target neurons. The vector contains a capsid that contains a mixture of capsid proteins from an AAV serotype such as AAV serotype 2 (retroAAV) (Tevro et al., Neuron 92:372-378 (2016), which is incorporated herein by reference) where point mutations increase retrograde uptake and capsid proteins from a different AAV serotype such as AAV serotype rh10. This capsid mixture produces a viral vector with significantly enhanced retrograde uptake and efficiency into afferent neurons compared to a capsid containing only proteins from retroAAV. The vector provides efficient control of organ function.

[0009] The present disclosure also provides a method for the regulatory control of organ function. The method generally comprises delivering a gene to the afferent neurons of an organ via a retrograde vector, where the product of the gene responds to an external drug or stimulus to control organ function. In one example, the method can be used to induce satiety and reduce food intake to control body weight. In this example, a retrograde AAV (retroAAV) expressing a designer receptor exclusively activated by designer drugs (DREADD) that activates neurons is injected into the gastric wall and taken up into the afferent vagal sensory neurons of the stomach, which respond to gastric distension and induce satiety. After systemic administration of a DREADD activator such as clozapine-N-oxide (CNO), satiety is induced and food intake is reduced. Other examples include delivering an excitatory chemogenetic ion channel to these neurons, followed by activation with an appropriate drug, or delivering an excitatory optogenetic ion channel ChR2, followed by delivery of light to the nerve fibers to activate ChR2. In another example, this method is used to control intractable cough not due to an underlying treatable disease. In one example, a retrograde AAV expressing an inhibitory DREADD is nebulized and inhaled to be taken up into the vagal sensory neurons of the lung, followed by systemic administration of a DREADD activator such as CNO to inhibit the activity of these sensory neurons and thereby reduce the cough reflex. In one example, a retrograde AAV expressing, for example, an inhibitory DREADD is injected into, for example, the vagus nerve or the nodose ganglion, followed by systemic administration of a DREADD activator such as CNO to inhibit the activity of these sensory neurons and thereby reduce the cough reflex.

[0010] In one embodiment, the viral vector encodes hM4Di, which is an engineered version of the M4 muscarinic acetylcholine receptor and is activated by CNO, clozapine, perlapine, or compound 21 (see Chen et al., ACS Chem.Neurosic., 6:476(2015)) (which is incorporated herein by reference) causes membrane hyperpolarization when combined with it by reducing cAMP signaling and increasing the activation of inwardly rectifying potassium channels. This produces a transient inhibition of neuronal activity similar to that seen after endogenous activation of the M4 receptor. hM3Dq (hD3q) is an engineered version of the M3 muscarinic receptor that, when activated by CNO, causes activation of the phospholipase C cascade, alters intracellular calcium, and results in burst-like firing of neurons. rM3D causes neuronal depolarization based on G-protein signaling (e.g., increased cAMP), which can regulate neuronal activity through a protein-based inhibitory signaling process rather than G-protein signaling. Other options for neuronal excitation are other rM3D, which similarly cause neuronal depolarization based on G-protein signaling (e.g., increased cAMP) (see, e.g., Dong, Allen, Farrell, and Roth, 2010; Ferguson, Phillips, Roth, Wess, and Neumaier, 2013); and Rq (R165L), which can regulate neuronal activity through a protein-based inhibitory signaling process rather than G-protein signaling. Another receptor is the inhibitory DREADD receptor Pdi. A mutant form of the Gi-coupled κ-opioid receptor (KORD) is activated by salvinorin B (SalB) and can therefore also be used for viral vectors.

[0011] The present disclosure also provides a method of preventing the spread of toxic proteins from the gastrointestinal tract to the brain by transferring a gene that prevents the transfer of toxic proteins to the vagus nerve. This can be done by directly injecting a viral vector into the sensory ganglia of the vagus nerve (such as the nodose ganglion), or directly injecting a viral vector into the vagal efferent cell bodies in the brain (such as the dorsal motor nucleus of the vagus nerve), or by injecting a viral vector into the gastrointestinal wall or by oral administration, and then these vectors are taken up into the vagal axons and transported retrogradely to express a therapeutic agent in the cell bodies. In one example, shRNA against α-synuclein (which prevents the expression of α-synuclein in target neurons) is expressed from a retrograde form of a virus such as a retroAAV / rh10 vector, and is delivered to the vagal sensory fibers by injection into the wall of the stomach and / or intestine. The ultimate expression of shRNA in the vagal sensory neurons blocks the expression of endogenous α-synuclein in these neurons, thereby preventing the spread of toxic synucleinopathy from the pathological fibrils in the gastrointestinal tract, resulting in the widespread brain pathology observed in Parkinson's disease, because the propagation of pathological synuclein requires expression in neurons. In another example, an antibody against α-synuclein is expressed in the vagal sensory fibers delivered by the gastrointestinal tract by a retroAAV / rh10 vector to prevent the spread of α-synuclein. In one embodiment, the rAAV has a capsid with at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity to SEQ ID NO:5 or 7. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Depiction of the expression of fluorescent mCherry protein in the afferent (sensory) fibers in the dorsal motor nucleus region of the vagus nerve in the brain after injection of an AAV vector into the stomach wall. A known retrograde tracer (cholera toxin subunit B) was injected into the stomach to label the afferent motor fibers. Injection of a mixed vector of AAV serotypes 2 and 1 (AAV2 / 1) showed virtually no neuronal uptake. Injection of retroAAV (showing increased retrograde uptake in the brain) caused expression of the mCherry protein in the neuronal fibers of the dorsal motor nucleus, rather than in the cell bodies, indicating that these are sensory fibers projecting from the nodose ganglion. The same procedure with a retroAAV and AAVrh10 chimeric capsid showed an increase in the number of fibers expressing mCherry in the same brain region.

[0013] Figure 2ADepicts selective expression of mCherry protein within the nodose ganglion following injection of AAV vector into the stomach wall. The nodose ganglion provides cell bodies for vagal sensory neurons. Cholera toxin tracer shows labeling of a small number of neuronal cell bodies within the nodose ganglion. In fact, no positive cell bodies were observed in the case of AAV2 / 1, but we observed some retrograde uptake into neurons in the brain. RetroAAV shows an increase in the number of cell bodies within the nodose ganglion expressing mCherry, indicating efficient retrograde uptake, but this uptake is selective as only a subset of nodose neurons are labeled. Compared to retroAAV alone, the retroAAV / rh10 hybrid vector demonstrates more labeled nodose neuronal cell bodies, but these still represent a subset of neurons( Figure 2B ). Quantification of neuronal cell counts shows that the number of positive neurons within the nodose ganglion increased approximately 3-fold following administration of retroAAV / rh10 to the stomach wall compared to retroAAV alone.

[0014] Figure 3 Depicts the response of fasted mice with normal food intake to 1 mg / kg CNO following injection of retroAAV / rh10 into the stomach wall. RetroAAV / rh10 expresses the DREADD (hD3q) that activates neurons. Animals injected with retroAAV / rh10 expressing the marker gene mCherry had normal food intake in response to CNO. Animals injected with retroAAV / rh10 expressing DREADD but given saline instead of CNO had the same normal food intake at 6 hours and 24 hours after administration of saline compared to the mCherry animals given CNO. Animals injected with retroAAV / rh10 expressing DREADD and then given CNO showed a significant reduction in food intake within hours after CNO administration. When CNO was cleared, the feeding behavior returned to normal, with a reduction in total food intake within 24 hours compared to the control, as food intake decreased within 4 - 6 hours after CNO and showed food intake similar to the control at that time within 6 - 24 hours after CNO was cleared.

[0015] Figure 4A - 4B Depicts the response of mice injected with retroAAV / rh10 expressing DREADD (hD3q) into the stomach wall and fasted for 24 hours before administration of CNO or saline. Compared to mice with normal food intake ( Figure 3In contrast, animals injected with retroAAV / rh10 expressing the marker gene mCherry had increased feeding at 6 and 24 hours after administration of CNO. Animals injected with retroAAV / rh10 expressing DREADD but given saline instead of CNO had the same feeding at 6 and 24 hours after administration of saline as the mCherry animals given CNO. Although the animals had been starved for the previous 24 hours, within a few hours after administration of CNO, animals injected with retroAAV / rh10 expressing DREADD and given CNO showed a significant reduction in food intake. This effect was so profound that within 4 - 6 hours after administration of CNO, the food intake of this starved group was still lower than that of animals that had normal feeding before the test and then received a control vehicle or drug (see Figure 3 ). When CNO was cleared, the feeding behavior of this group rebounded compared to the normal feeding group, and the food intake of the animals increased at 6 - 24 hours after CNO was cleared, because the starvation period was effectively prolonged by the treatment compared to the control. This is different from normal - feeding mice, which resumed normal food intake after CNO was cleared but did not increase intake compared to the control.

[0016] Figure 5 Exemplary pathways for delivering gene therapy to control organ function are shown.

[0017] Figure 6 Data depicting the feeding behavior of fasted mice at 3 mg / kg CNO.

[0018] Figure 7 Data showing the feeding behavior of normal - feeding mice at 3 mg / kg CNO.

[0019] Figure 8A - 8E Data depicting the feeding behavior of normal - feeding mice at 1 mg / kg CNO.

[0020] Figure 9A - 9E Showing the long - term stability of reduced feeding behavior in normal - feeding mice at 1 mg / kg CNO.

[0021] Figure 10 Showing a reduced increase in body weight in gut retro / rh10AAV HD3q (DREADD) mice on a 60% high - fat diet treated with 1 mg / kg CNO daily (C) compared to saline (S).

[0022] Figure 11Depicts the continuous reduction in body weight gain in intestinal retro / rh10AAV HD3q (DREADD) mice on a 60% high-fat diet treated with 1 mg / kg CNO (C) per day compared to saline (S). The X-axis shows the sequential days after the start of drug treatment, and day 33 was day 24 in the previous figure.

[0023] Figure 12A - 12B Shows the sequence of pNLRep2_RETRO Cap2 (SEQ ID NO:1).

[0024] Figure 13A - 13U Provides the sequence of pNLRep2_rh10Cap (SEQ ID NO:2).

[0025] Figure 14A - 14B Shows the sequence of AAV.CBA.flag-mCheryy.WPRE (SEQ ID NO:3).

[0026] Figure 15A - 15P Provides exemplary sequences of the AAVrh10 (SEQ ID No.7) and retroAAV2 (SEQ ID No.5) capsids. A portion of SEQ ID NO:4 encodes retroAAV2. A portion of SEQ ID NO:6 encodes AAVrh10.

[0027] Figure 16A - 16B Provides the sequences of human M3 and M4. The DREADD of hM3 can have substitutions at residues 149 and / or 239, for example, Y149C or A239G in mM3, and the DREADDS of hM4 can have substitutions at residues 113 and / or 203, for example, Y113C or A203G in mM4 (SEQ ID NO:12). Detailed Description

[0028] Definitions

[0029] "Mediator" refers to a polynucleotide or a macromolecule or macromolecule-related entity that includes a polynucleotide and is related to a polynucleotide and can be used to mediate the delivery of a polynucleotide to a cell in vitro or in vivo. Exemplary mediators include, for example, plasmids, viral mediators, liposomes, and other gene delivery mediators. The polynucleotide to be delivered, sometimes referred to as the "target polynucleotide" or "transgene", can include a target coding sequence in a gene polypeptide or peptide suitable for eliciting an immune response in a mammal, and / or a selectable or detectable marker.

[0030] As used herein, "transduction", "transfection", "transformation" or "transducing" refers to the process of introducing an exogenous polynucleotide into a host cell, thereby causing the expression of a polynucleotide, such as a transgene, in the cell, and includes the use of a recombinant virus to introduce the exogenous polynucleotide into the host cell. Transduction, transfection or transformation of a polynucleotide in a cell can be determined by methods well known in the art, including but not limited to measuring protein expression (including steady-state levels), such as by ELISA, flow cytometry and Western blotting, and measuring DNA and RNA by hybridization assays, such as Northern blotting, Southern blotting and gel mobility shift assays. Methods for introducing exogenous polynucleotides include well-known techniques such as viral infection or transfection, lipofection, transformation and electroporation, as well as other non-viral gene delivery techniques. The introduced polynucleotide can be maintained stably or transiently in the host cell.

[0031] "Gene delivery" refers to the introduction of an exogenous polynucleotide into a cell for gene transfer and can encompass targeting, binding, uptake, transport, localization, replicon integration and expression.

[0032] "Gene transfer" refers to the introduction of an exogenous polynucleotide into a cell, which can encompass targeting, binding, uptake, transport, localization and replicon integration, but is distinct from and does not imply subsequent expression of the gene.

[0033] "Gene expression" or "expression" refers to the process of gene transcription, translation and post-translational modification.

[0034] An "infectious" virus or viral particle is a virus or viral particle that includes a polynucleotide component and is capable of delivering the polynucleotide component to a cell that provides sustenance for the viral species. The term does not necessarily imply any replication ability of the virus.

[0035] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides or their analogs. Polynucleotides can include modified nucleotides, such as methylated or capped nucleotides and nucleotide analogs, and can be interrupted by non-nucleotide components. If modifications are present, the nucleotide structure can be modified either before or after polymer assembly. The term polynucleotide as used herein refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise stated or required, any embodiment of the invention described herein regarding polynucleotides encompasses each of the double-stranded form and the two complementary single-stranded forms known or predicted to constitute the double-stranded form.

[0036] "Isolated" polynucleotides, such as plasmids, viruses, polypeptides or other substances, refer to a preparation of such substances that is free of at least some other components that may also be present where the substance or similar substances are found in nature or were initially prepared. Thus, for example, an isolated substance can be prepared by enriching the substance from a source mixture using purification techniques. Isolated nucleic acids, peptides or polypeptides exist in a form or environment different from that found in nature. For example, a given DNA sequence (e.g., a gene) is found on the host cell chromosome adjacent to neighboring genes; an RNA sequence, such as a particular mRNA sequence encoding a specific protein, is found in a cell as a mixture with many other mRNAs encoding many proteins. Isolated nucleic acid molecules can exist in single-stranded or double-stranded form. When an isolated nucleic acid molecule is used to express a protein, the molecule will contain at least the sense strand or coding strand (i.e., the molecule can be single-stranded), but can contain both the sense strand and the antisense strand (i.e., the molecule can be double-stranded). Enrichment can be measured on an absolute basis, such as weight per volume of solution, or it can be measured relative to a second potentially interfering substance present in the source mixture. Embodiments of the present invention are increasingly enriched, the more preferred. Thus, for example, 2-fold enrichment, 10-fold enrichment, 100-fold enrichment or 1000-fold enrichment.

[0037] "Transcription regulatory sequence" refers to a genomic region that controls the transcription of a gene or coding sequence operably linked thereto. Transcription regulatory sequences for use in the present invention typically comprise at least one transcription promoter and may also comprise one or more transcription enhancers and / or transcription terminators.

[0038] "Operably linked" refers to the arrangement of two or more components in which the components so described are in a relationship that permits them to operate in a coordinated manner. By way of example, a transcription regulatory sequence or promoter is operably linked to a coding sequence if the TRS or promoter facilitates the transcription of the coding sequence. An operably linked TRS is typically cis-linked to the coding sequence, but it need not be directly adjacent to the coding sequence.

[0039] "Heterologous" means derived from an entity that is genotypically different from the entity with which it is being compared. For example, a polynucleotide introduced into a different cell type by genetic engineering techniques is a heterologous polynucleotide (and when expressed, can encode a heterologous polypeptide). Similarly, a transcription regulatory element such as a promoter that has been removed from its native coding sequence and operably linked to a different coding sequence is a heterologous transcription regulatory element.

[0040] "Terminator" refers to a polynucleotide sequence that tends to reduce or prevent readthrough transcription (i.e., it reduces or prevents transcription initiated on one side of the terminator from continuing to the other side of the terminator). The extent to which transcription is disrupted typically varies with the base sequence and / or the length of the terminator sequence. Specifically, as is well known in many molecular biology systems, certain DNA sequences commonly referred to as "transcription termination sequences" are specific sequences that tend to disrupt readthrough transcription by RNA polymerase, presumably by causing the RNA polymerase molecule to stop and / or dissociate from the DNA being transcribed. Typical examples of such sequence-specific terminators include polyadenylation ("polyA") sequences, such as the SV40 polyA. In addition to or instead of such sequence-specific terminators, insertion of a relatively long DNA sequence between the promoter and the coding region also tends to disrupt transcription of the coding region, typically in proportion to the length of the inserted sequence. This effect may be because RNA polymerase molecules always have some tendency to dissociate from the DNA being transcribed to some extent, and increasing the length of the sequence to be traversed before reaching the coding region will generally increase the likelihood of dissociation occurring before transcription of the coding region is completed or even possibly before it begins. Thus, a terminator can prevent transcription from only one direction ("unidirectional" terminator) or from both directions ("bidirectional" terminator), and can consist of a sequence-specific termination sequence or a sequence-nonspecific terminator or both. Various such terminator sequences are known in the art; and illustrative uses of these sequences in the context of the present invention are provided below.

[0041] "Host cell", "cell line", "cell culture", "packaging cell line" and other such terms denote higher eukaryotic cells that can be used in the present invention, for example, to produce recombinant viruses or recombinant fusion polypeptides, such as mammalian cells including human cells. These cells include the progeny of the original cells that have been transduced. It should be understood that the progeny of a single cell may not necessarily be identical to the original parental cell (in terms of morphology or genomic complement).

[0042] "Recombinant" as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction and / or ligation steps and other procedures that result in a construct different from the polynucleotide found in nature. A recombinant virus is a viral particle that includes a recombinant polynucleotide. These terms respectively include the replication of the original polynucleotide construct and the progeny of the original viral construct.

[0043] "Control element" or "control sequence" refers to a nucleotide sequence that participates in molecular interactions and contributes to the regulation of the function of a polynucleotide, including replication, reiteration, transcription, splicing, translation, or degradation of the polynucleotide. Such regulation may affect the frequency, rate, or specificity of the process and may be either enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region that, under certain conditions, is capable of binding RNA polymerase and initiating transcription of a coding region, usually located downstream (in the 3' direction) of the promoter. Promoters include AAV promoters such as P5, P19, P40, and the AAV ITR promoter, as well as heterologous promoters.

[0044] "Expression vector" refers to a vector that includes a region encoding a target gene product and is used to effect the expression of the gene product in a desired target cell. The expression vector also includes control elements operably linked to the coding region to facilitate the expression of the protein in the target. The combination of control elements and one or more genes to which they are operably linked for expression is sometimes referred to as an "expression cassette", many of which are known and available in the art or can be readily constructed from components available in the art.

[0045] The terms "polypeptide" and "protein" are used interchangeably herein to refer to an amino acid polymer of any length. The terms also encompass amino acid polymers that have been modified; for example, disulfide bond formation, glycosylation, acetylation, phosphorylation, lipidation, or conjugation to a labeling component.

[0046] The term "exogenous", when used in connection with a protein, gene, nucleic acid, or polynucleotide in a cell or organism, refers to a protein, gene, nucleic acid, or polynucleotide that has been introduced into the cell or organism by artificial or natural means. An exogenous nucleic acid can be from a different organism or cell, or it can be one or more additional copies of a nucleic acid that is naturally present in the organism or cell. As a non-limiting example, an exogenous nucleic acid is located at a chromosomal position different from that of the native cell or is flanked by nucleic acid sequences different from those found in nature; for example, an expression cassette that ligates a promoter from one gene to the open reading frame of the gene product of a different gene.

[0047] "Transformed" or "transgenic" is used herein to include any host cell or cell line that has been altered or amplified due to the presence of at least one recombinant DNA sequence. The host cells of the present invention are typically produced by transfection with a DNA sequence in a plasmid expression vector, as an isolated linear DNA sequence, or by infection with a recombinant viral vector.

[0048] The term "sequence homology" means the proportion of base matches between two nucleic acid sequences or the proportion of amino acid matches between two amino acid sequences. When sequence homology is expressed as a percentage, e.g., 50%, the percentage represents the proportion of matches over the length of the selected sequence compared to other sequences. Gaps (in either of the two sequences) are allowed to maximize the matches; generally, a gap length of 15 bases or less is used, preferably 6 bases or less, more preferably 2 bases or less. When using oligonucleotides as probes or therapeutics, the sequence homology between the target nucleic acid and the oligonucleotide sequence is generally as follows: no fewer than 17 target base matches (85%) out of 20 possible oligonucleotide base pair matches; no fewer than 9 matches (90%) out of 10 possible base pair matches; or no fewer than 19 matches (95%) out of 20 possible base pair matches.

[0049] Two amino acid sequences are homologous if there is partial or complete identity between them. For example, 85% homology means that when the two sequences are aligned for maximum matching, 85% of the amino acids are identical. Gaps (in either of the two matching sequences) are allowed when maximizing the matches; preferably a gap length of 5 or less, more preferably 2 or less. Alternatively and preferably, if the program ALIGN with a mutation data matrix and a gap penalty of 6 or greater is used, two protein sequences (or polypeptide sequences of at least 30 amino acids in length derived from them) are homologous (as the term is used herein) if they have an alignment score greater than 5 (in standard deviation units). When using the ALIGN program for optimal alignment, two sequences or parts thereof are more homologous if their amino acids are 50% or more identical.

[0050] The term "corresponds to" is used herein to mean that a polynucleotide sequence is structurally related to all or part of a reference polynucleotide sequence, or a polypeptide sequence is structurally related to all or part of a reference polypeptide sequence, e.g., they have at least 80%, 85%, 90%, 95% or more, e.g., 99% or 100% sequence identity. In contrast, the term "complementary" is used herein to mean that a complementary sequence is homologous to all or part of a reference polynucleotide sequence. By way of example, the nucleotide sequence "TATAC" corresponds to the reference sequence "TATAC" and is complementary to the reference sequence "GTATA".

[0051] The term "sequence identity" means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) within a comparison window. The term "percent sequence identity" means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) within a comparison window. The term "percent sequence identity" is calculated by comparing two optimally aligned sequences within a comparison window, determining the number of positions at which the identical nucleic acid bases (e.g., A, T, C, G, U, or I) occur in both sequences to yield the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to yield the percent sequence identity. The term "substantially identical" as used herein denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence having at least 85% sequence identity, preferably at least 90% to 95% sequence identity, more typically at least 99% sequence identity compared to a reference sequence within a comparison window of at least 20 nucleotide positions, usually in a window of at least 20 - 50 nucleotides, wherein the percent sequence identity is calculated by comparing the reference sequence with the polynucleotide sequence, which may include deletions or additions totaling up to 20% of the reference sequence within the comparison window.

[0052] "Conservative" amino acid substitutions are, for example, aspartic acid - glutamic acid as polar acidic amino acids; lysine / arginine / histidine as polar basic amino acids; leucine / isoleucine / methionine / valine / alanine / glycine / proline as non - polar or hydrophobic amino acids; serine / threonine as polar or uncharged hydrophilic amino acids. Conservative amino acid substitutions also include groupings based on side chains. For example, a group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids with aliphatic hydroxyl side chains is serine and threonine; a group of amino acids with amide - containing side chains is asparagine and glutamine; a group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids with basic side chains is lysine, arginine, and histidine; and a group of amino acids with sulfur - containing side chains is cysteine and methionine. For example, it can be reasonably expected that replacing leucine with isoleucine or valine, aspartic acid with glutamic acid, threonine with serine, or replacing an amino acid with a structurally related amino acid analog will not have a significant impact on the properties of the resulting polypeptide. Whether an amino acid change results in a functional polypeptide can be easily determined by measuring the specific activity of the polypeptide. Based on common side - chain properties, naturally occurring residues are grouped into several categories: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues affecting chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe.

[0053] The present disclosure also contemplates polypeptides with non - conservative substitutions. Non - conservative substitutions require replacing a member of one of the above categories with another.

[0054] Gene transfer mediator

[0055] The present disclosure provides a gene transfer mediator, such as a viral gene transfer mediator, which can be used to deliver genes to neurons or nerve fibers, or to disperse toxic gene products (such as toxic proteins) from the gastrointestinal tract to the brain. Various aspects of gene transfer mediators and methods are discussed below. Thus, according to the gene transfer mediators and methods, any combination of parameters can be used.

[0056] A "gene transfer mediator" is any molecule or composition that is capable of carrying a heterologous nucleic acid sequence to a suitable host cell in which encoding protein synthesis occurs. Typically, the gene transfer mediator is a nucleic acid molecule that has been engineered using recombinant DNA techniques known in the art to incorporate a heterologous nucleic acid sequence. Desirably, the gene transfer mediator is composed of DNA. Examples of suitable DNA-based gene transfer mediators include plasmids and viral mediators. However, non-nucleic acid-based gene transfer mediators, such as liposomes, are also known and used in the art. The gene transfer mediators of the present invention can be based on a single type of nucleic acid (e.g., plasmid) or non-nucleic acid molecule (e.g., lipid or polymer). The gene transfer mediator can integrate into the host cell genome or can exist episomally in the host cell.

[0057] In one embodiment, the gene transfer mediator is a viral mediator. Suitable viral mediators include, for example, retroviral mediators, herpes simplex virus (HSV)-based mediators, parvovirus-based mediators, such as adeno-associated virus (AAV)-based mediators, AAV-adenovirus chimeric mediators, and adenovirus-based mediators. These viral mediators can be prepared using standard recombinant DNA techniques described, for example, in Sambrook et al., Molecular Cloning, a Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, New York, N.Y. (1994).

[0058] In one embodiment, the present invention provides an adeno-associated virus (AAV) vector. The AAV vector can contain a gene to be expressed and additional components that do not substantially affect the AAV vector (e.g., genetic elements such as a poly(A) sequence or a restriction enzyme site that facilitate in vitro manipulation of the vector). Adeno-associated virus is a member of the parvovirus family and includes a linear single-stranded DNA genome of less than about 5,000 nucleotides. Co-infection with a helper virus (i.e., adenovirus or herpesvirus) or expression of helper genes is required for efficient AAV replication. AAV vectors for the administration of therapeutic nucleic acids typically have approximately 96% of the parental genome deleted, leaving only the inverted terminal repeats (ITRs) that contain the recognition signals for DNA replication and packaging. This eliminates immune or toxic side effects caused by viral gene expression. Additionally, when needed, delivery of specific AAV proteins to producer cells can integrate the AAV vector, including the AAV ITRs, into specific regions of the cellular genome (see, e.g., U.S. Patent Nos. 6,342,390 and 6,821,511). Host cells containing the integrated AAV genome do not change in cell growth or morphology (see, e.g., U.S. Patent No. 4,797,368).

[0059] The AAV ITRs flank the unique coding nucleotide sequences of the non-structural replication (Rep) proteins and the structural capsid (Cap) proteins (also known as virion proteins (VPs)). The terminal 145 nucleotides are self-complementary and are organized such that an energetically stable intramolecular duplex that forms a T-shaped hairpin can be formed. These hairpin structures serve as the origin of viral DNA replication by acting as primers for the cellular DNA polymerase complex. The Rep gene encodes the Rep proteins Rep78, Rep68, Rep52, and Rep40. Rep78 and Rep68 are transcribed from the p5 promoter, and Rep52 and Rep40 are transcribed from the p19 promoter. The Rep78 and Rep68 proteins are multifunctional DNA-binding proteins that perform helicase and nicking enzyme functions during productive replication to allow for the excision of the AAV termini (see, e.g., Im et al., Cell , 61 :447(1990)). These proteins also regulate the transcription of the endogenous AAV promoter and promoters within helper viruses (see, e.g., Pereira et al., J.Virol. , 71 :1079(1997)). The other Rep proteins modify the functions of Rep78 and Rep68. The cap gene encodes the capsid proteins VP1, VP2, and VP3. The cap gene is transcribed from the p40 promoter.

[0060] Any AAV serotype known in the art can be used to generate AAV vectors. Several AAV serotypes and over 100 AAV variants have been isolated from adenovirus stocks or from human or non-human primate tissues (reviewed in, for example, Wu et al., Molecular Therapy , 14 (3):316(2006)). Generally, AAV serotypes have genomic sequences that are significantly homologous at the nucleic acid sequence and amino acid sequence levels, such that different serotypes have the same set of genetic functions, produce virions that are physically and functionally substantially equivalent, and replicate and assemble by virtually the same mechanisms. AAV serotypes 1-6 and 7-9 are defined as "true" serotypes because they do not cross-react efficiently with neutralizing sera that are specific for all other existing and characterized serotypes. In contrast, AAV serotypes 6, 10 (also known as Rh10), and 11 are considered "variant" serotypes because they do not meet the definition of "true" serotypes. AAV serotype 2 (AAV2) has been widely used in gene therapy applications due to its lack of pathogenicity, broad tropism, and ability to establish long-term transgene expression (see, for example, Carter, Hum.Gene Ther. , 16 :541(2005); and Wu et al. (supra)). The genomic sequences of various AAV serotypes and their comparisons are publicly available, for example, in GenBank accession numbers U89790, J01901, AF043303, and AF085716; Chiorini et al., J.Virol. , 71 :6823(1997); Srivastava et al., J.Virol. , 45 :555(1983); Chiorini et al., J.Virol. , 73 :1309(1999); Rutledge et al., J.Virol. , 72 :309(1998); and Wu et al., J.Virol. , 74 :8635(2000).

[0061] The AAV rep and ITR sequences are particularly conserved in most AAV serotypes. For example, the Rep78 proteins of AAV2, AAV3A, AAV3B, AAV4, and AAV6 are reported to be approximately 89%-93% identical (see Bantel-Schaal et al., J.Virol. , 73(2):939(1999)). It has been reported that AAV serotypes 2, 3A, 3B, and 6 share approximately 82% total nucleotide sequence identity at the genomic level (Bantel-Schaal et al. (supra)). In addition, the rep sequences and ITRs of many AAV serotypes are known to cross-complement (e.g., functionally replace) corresponding sequences from other serotypes efficiently during the production of AAV particles in mammalian cells.

[0062] Generally, the cap proteins that determine the tropism of AAV particles and the associated cap protein-encoding sequences are significantly less conserved among different AAV serotypes than the Rep gene. Given the ability of Rep and ITR sequences to cross-complement corresponding sequences of other serotypes, AAV vectors can include mixtures of serotypes and are thus "chimeric" or "pseudotyped" AAV vectors. Chimeric AAV vectors typically include AAV capsid proteins derived from two or more (e.g., 2, 3, 4, etc.) different AAV serotypes. In contrast, pseudotyped AAV vectors include one or more ITRs of one AAV serotype packaged in the capsid of another AAV serotype. Chimeric and pseudotyped AAV vectors are further described in, for example, U.S. Patent No. 6,723,551; Flotte, Mol.Ther. , 13 (1):1(2006); Gao et al., J.Virol. , 78 :6381(2004); Gao et al., Proc.Natl.Acad.Sci.USA , 99 :11854(2002); De et al., Mol.Ther. , 13 :67(2006); and Gao et al., Mol.Ther. , 13 :77(2006).

[0063] In one embodiment, the AAV vector is produced using AAV that infects humans (e.g., AAV2). Alternatively, the AAV vector is produced using AAV that infects non-human primates (e.g., anthropoids (e.g., chimpanzees), Old World monkeys (e.g., macaques), and New World monkeys (e.g., marmosets)). In one embodiment, the AAV vector is produced using AAV that infects non-human primates pseudotyped with AAV that infects humans. Examples of such pseudotyped AAV vectors are disclosed in, for example, Cearley et al., Molecular Therapy , 13:528(2006). In one embodiment, an AAV vector can be generated that comprises a capsid protein from AAV that infects rhesus monkeys pseudotyped with AAV2 inverted terminal repeats (ITRs). In certain embodiments, the AAV vectors of the invention comprise capsid proteins from AAV10 (also referred to as "AAVrh.10") that infects rhesus monkeys pseudotyped with AAV2 ITRs (see, e.g., Watanabe et al., Gene Ther. , 17 (8):1042(2010); and Mao et al., Hum.Gene Therapy , 22 :1525(2011)).

[0064] In addition to the gene to be expressed, the AAV vector can also include expression control sequences such as promoters, enhancers, polyadenylation signals, transcription terminators, internal ribosome entry sites (IRES), etc., which provide for the expression of the nucleic acid sequence in a host cell. Exemplary expression control sequences are known in the art and are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, CA. (1990).

[0065] A large number of promoters from a variety of different sources, including constitutive, inducible, and repressible promoters, are well known in the art. Representative sources of promoters include, for example, viruses, mammals, insects, plants, yeast, and bacteria, and suitable promoters from these sources are readily available or can be synthesized based on sequences publicly available from, for example, depository centers such as ATCC and other commercial or individual sources. A promoter can be unidirectional (i.e., initiate transcription in one direction) or bidirectional (i.e., initiate transcription in the 3' or 5' direction). Non-limiting examples of promoters include, for example, the T7 bacterial expression system, the pBAD(araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter, and the RSV promoter. Inducible promoters include, for example, the Tet system (U.S. Patent Nos. 5,464,758 and 5,814,618), the ecdysone-inducible system (No et al., Proc.Natl.Acad.Sci. , 93 :3346(1996)), the T-REXTM system (Invitrogen, Carlsbad, CA), the LACSWITCH TM system (Stratagene, San Diego, CA), and the Cre-ERT tamoxifen-inducible recombinase system (Indra et al.,Nuc.Acid.Res. , 27 : 4324 (1999); Nuc.Acid.Res. , 28 : e99 (2000); U.S. Patent No. 7,112,715; and Kramer and Fussenegger, Methods Mol.Biol. , 308 : 123 (2005)).

[0066] As used herein, the term "enhancer" refers to a DNA sequence that increases the transcription of, for example, a nucleic acid sequence operably linked thereto. Enhancers can be located thousands of bases away from the coding region of the nucleic acid sequence and can mediate the binding of regulatory factors, patterns of DNA methylation, or changes in DNA structure. A large number of enhancers from various different sources are well known in the art and can be obtained as cloned polynucleotides or within cloned polynucleotides (from, for example, depository centers such as ATCC and other commercial or individual sources). Many polynucleotides that include promoters (such as the commonly used CMV promoter) also include enhancer sequences. Enhancers can be located upstream, within, or downstream of the coding sequence. In one embodiment, the nucleic acid sequence is operably linked to the CMV enhancer / chicken β-actin promoter (also referred to as the "CAG promoter") (see, for example, Niwa et al., Gene , 108 : 193 (1991); Daly et al., Proc.Natl.Acad.Sci.U.S.A. , 96 : 2296 (1999); and Sondhi et al., Mol.Ther. , 15 : 481 (2007)).

[0067] Typically, AAV vectors are produced using well-characterized plasmids. For example, human embryonic kidney 293T cells are transfected with a transgene-specific plasmid and another plasmid containing adenovirus helper genes and AAV rep and cap genes (specific for AAVrh.10, 8, or 9 as needed). After 72 hours, the cells are harvested and the vector is released from the cells by five freeze / thaw cycles. Subsequent centrifugation and benzonase treatment remove cell debris and DNA not encapsulated in the capsid. Iodixanol gradients and ion exchange columns can be used to further purify each AAV vector. Next, the purified vector is concentrated to the desired concentration by size exclusion centrifugal spin columns. Finally, the buffer is exchanged to produce the final vector product formulated in (for example) 1x phosphate-buffered saline. The virus titer can be measured by real-time PCR, and the virus purity can be evaluated by SDS-PAGE.

[0068] Drug compositions and delivery

[0069] The present invention provides a composition which comprises the above gene transfer mediator and a pharmaceutically acceptable (e.g., physiologically acceptable) carrier, consists essentially of these components or consists of these components. When the composition consists essentially of the gene transfer mediator of the present invention and a pharmaceutically acceptable carrier, additional components (e.g., adjuvants, buffers, stabilizers, anti-inflammatory agents, solubilizers, preservatives, etc.) that do not substantially affect the composition may be included. When the composition consists of the gene transfer mediator of the present invention and a pharmaceutically acceptable carrier, the composition does not include any additional components. Any suitable carrier can be used in the context of the present invention, and such carriers are well known in the art. The choice of carrier will be determined in part by the particular site to which the composition may be administered and the particular method for administering the composition. In addition to the gene transfer mediator described herein, the composition may optionally be sterile. The composition can be stored frozen or lyophilized and reconstituted in a suitable sterile carrier prior to use. The composition can be produced according to conventional techniques described, for example, in Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).

[0070] Formulations suitable for the composition include aqueous and non-aqueous solutions, an isotonic sterile solution that may contain antioxidants, buffers, and bacteriostatic agents, and aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations may be presented in unit-dose or multi-dose sealed containers such as ampoules and vials and may be stored under lyophilized (freeze-dried) conditions, requiring only the addition of a sterile liquid carrier, such as water, immediately prior to use. The temporary solutions and suspensions may be prepared from sterile powders, granules, and tablets of the above types. In one embodiment, the carrier is a buffered saline solution. In one embodiment, the gene transfer mediator of the present invention is administered in the form of a composition that is formulated to protect the gene transfer mediator from destruction prior to administration. For example, the composition may be formulated to reduce losses of the gene transfer mediator on devices used for preparing, storing, or administering the gene transfer mediator, such as glassware, syringes, or needles. The composition may be formulated to reduce the light sensitivity and / or temperature sensitivity of the gene transfer mediator. To this end, the composition may include a pharmaceutically acceptable liquid carrier, such as those described above, and a stabilizer selected from the group consisting of polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, and combinations thereof. Using such a composition will extend the shelf life of the gene transfer mediator, facilitate administration, and increase the efficiency of the methods of the present invention. Formulations of compositions containing a gene transfer mediator are further described, for example, in Wright et al., Curr. Opin. Drug Discov. Devel., 6(2):174-178 (2003) and Wright et al., Molecular Therapy, 12:171-178 (2005)).

[0071] The composition may also be formulated to enhance transduction efficiency. In addition, those of ordinary skill in the art will understand that the gene transfer mediator of the present invention may be present in the composition together with other therapeutic or bioactive agents. For example, factors that control inflammation, such as ibuprofen or steroids, may be part of the composition to reduce swelling and inflammation associated with in vivo administration of the gene transfer mediator. Immune system stimulants or adjuvants, such as interleukins, lipopolysaccharides, and double-stranded RNA, may be used to enhance or alter the immune response. Antibiotics, namely microbicides and fungicides, may be used to treat existing infections and / or reduce the risk of future infections, such as those associated with the gene transfer process.

[0072] The injectable long-acting form is made by forming a microcapsule matrix of the subject compound in a biodegradable polymer such as poly(lactide-co-glycolide). The rate of drug release can be controlled according to the ratio of the drug to the polymer and the nature of the particular polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Long-acting injectable formulations are also prepared by encapsulating the drug in liposomes or microemulsions that are compatible with body tissues.

[0073] In certain embodiments, the formulations of the present invention include biocompatible polymers selected from the group consisting of: polyamides, polycarbonates, polyalkylenes, polymers of acrylates and methacrylates, polyethylene polymers, polyglycolides, polysiloxanes, polyurethanes and their copolymers, celluloses, polypropylenes, polyethylenes, polystyrenes, polymers of lactic acid and glycolic acid, polyanhydrides, poly(orthoacids) esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), polysaccharides, proteins, polyhyaluronic acid, polycyanoacrylates and their blends, mixtures or copolymers.

[0074] The compositions can be administered in or on a device that allows controlled or sustained release, such as a sponge, a biocompatible mesh, a mechanical reservoir or a mechanical implant. Implants (see, for example, U.S. Patent No. 5,443,505), devices (see, for example, U.S. Patent No. 4,863,457) such as implantable devices (e.g., mechanical reservoirs or implants) or devices made of a polymeric composition are particularly useful for the administration of the gene transfer mediators of the present invention. The compositions can also be administered in the form of a sustained release formulation (see, for example, U.S. Patent No. 5,378,475), including, for example, gel foams, hyaluronic acid, gelatin, chondroitin sulfate, polyphosphates, such as bis-2-hydroxyethyl terephthalate (BHET) and / or poly(lactic-co-glycolic acid).

[0075] Delivery of the compositions comprising the gene transfer mediators can be by means known in the art, intracerebral (including but not limited to intracerebral parenchyma, intraventricular or intracisternal), intrathecal (including but not limited to lumbar or cisterna magna) or systemic, including but not limited to intravenous or any combination thereof. Delivery can also be by surgically implanting an implanted device.

[0076] The dose of the gene transfer mediator in the composition administered to a mammal will depend on many factors, including the size (mass) of the mammal, the degree of any side effects, the specific route of administration, etc. In one embodiment, the method of the present invention includes administering a "therapeutically effective amount" of a composition described herein comprising the gene transfer mediator of the present invention. A "therapeutically effective amount" refers to an effective amount that achieves the desired therapeutic effect at the necessary dose and time. The therapeutically effective amount can vary depending on factors such as the pathology, age, sex, and weight of the individual, as well as the ability of the gene transfer mediator to elicit the desired response in the individual. The dose of the gene transfer mediator in the composition required to achieve a particular therapeutic effect is typically administered in terms of the number of genomic copies of the mediator per cell (gc / cell) or the number of genomic copies of the mediator per kilogram of body weight (gc / kg). Those of ordinary skill in the art can readily determine an appropriate dose range of the gene transfer mediator based on these and other factors well known in the art to treat a patient suffering from a particular disease or disorder. The therapeutically effective amount can be between 1×10 10 genomic copies and 1×10 13 genomic copies.

[0077] In one embodiment, the mediator is an adenovirus, adeno-associated virus (AAV), retrovirus, or lentivirus mediator. In one embodiment, the AAV mediator is pseudotyped. In one embodiment, the AAV mediator is pseudotyped with the AAVrh.10, AAV8, AAV9, AAV5, AAVhu.37, AAVhu.20, AAVhu.43, AAVhu.8, AAVhu.2, or AAV7 capsid. In one embodiment, the AAV mediator is pseudotyped with the AAVrh.10, AAV8, or AAV5 capsid. In one embodiment, the AAV mediator is pseudotyped with the AAV2, AAV5, AAV7, AAV8, AAV9, or AAVrh.10 capsid. Further provided is a pharmaceutical composition comprising a certain amount of the above-described gene therapy mediator. The dose of the viral mediator can be from about 1×10 11 to about 1×10 16 genomic copies, from about 1×10 12 to about 1×10 15 genomic copies, from about 1×10 11 to about 1×10 13 genomic copies, or from about 1×10 13 to about 1×10 15 genomic copies.

[0078] In one embodiment of the present invention, the composition is administered once to a mammal. It is believed that a single administration of the composition will result in sustained expression in the mammal with minimal side effects. However, in certain cases, it may be appropriate to administer the composition multiple times during treatment to ensure sufficient exposure of the cells to the composition. For example, the composition may be administered to a mammal two or more times (e.g., 2, 3, 4, 5, 6, 6, 8, 9, or 10 times or more) during treatment.

[0079] Exemplary embodiments

[0080] The present disclosure provides materials and methods for controlling organ function to prevent, inhibit, or treat diseases. In one aspect, the method provides delivering a viral vector to an organ, and then the viral vector is taken up by axons that regulate the function of these organs. In one embodiment, the viral vector is an adeno-associated virus (AAV) vector. In one embodiment, a retrograde form of adeno-associated virus (AAV) is specifically taken up into a subset of vagal sensory neurons that respond to gastric distension and cause satiety when injected into the gastric wall. This represents a specific subset of neurons that project from the nodose ganglion without affecting other nodose neurons that provide sensation to other visceral organs. The viral vector can also be other forms of retrograde vectors, including but not limited to retrograde lentivirus (LV) vectors, herpes simplex virus (HSV) vectors, or canine adenovirus (CAV) vectors.

[0081] The present disclosure also provides a viral vector having improved properties for retrograde uptake into target neurons. This vector contains a capsid that contains a mixture of capsid proteins from an AAV serotype that has point mutations that increase retrograde uptake, such as AAV serotype 2 (retroAAV) (Tevro, et al., Neuron 92:372-378 (2016), which is incorporated herein by reference), and capsid proteins from a different AAV serotype, such as AAV serotype rh10. This capsid mixture produces a viral vector that has significantly enhanced retrograde uptake and efficiency into afferent neurons compared to a capsid that contains only proteins from retroAAV. This vector provides efficient control of organ function.

[0082] The present disclosure also provides a method for the regulatory control of organ function. The method generally comprises delivering a gene to the afferent neurons of an organ via a retrograde vector, the product of the gene responding to an external drug or stimulus to control organ function. In one example, this method can be used to induce satiety and reduce food intake to control body weight. In this example, a retrograde adeno-associated virus (retroAAV) expressing a designer receptor exclusively activated by designer drugs (DREADD) that activates neurons is injected into the gastric wall and taken up by the afferent vagal sensory neurons of the stomach, which respond to gastric distension and induce satiety. After systemic administration of a DREADD activator such as clozapine-N-oxide (CNO), satiety is induced and food intake is reduced. Other examples include delivering an excitatory chemogenetic ion channel to these neurons, followed by activation with an appropriate drug, or delivering an excitatory optogenetic ion channel, channelrhodopsin-2 (ChR2), followed by delivery of light to the nerve fibers to activate ChR2. In another example, this method is used to control intractable cough not caused by an underlying treatable disease. In this example, a retrograde AAV expressing an inhibitory DREADD is nebulized and inhaled to be taken up into the vagal sensory neurons of the lung, followed by systemic administration of a DREADD activator such as CNO to inhibit the activity of these sensory neurons and thereby reduce the cough reflex.

[0083] The present disclosure also provides a method for preventing the spread of toxic proteins from the gastrointestinal tract to the brain by transferring a gene that prevents the transfer of toxic proteins to the vagus nerve. This can be done by directly injecting a viral vector into the sensory ganglia of the vagus nerve (such as the nodose ganglion), or directly injecting a viral vector into the efferent cell bodies of the vagus nerve in the brain (such as the dorsal motor nucleus of the vagus nerve), or by injecting a viral vector into the gastrointestinal wall or by oral administration, and then these vectors are taken up into the vagus nerve axons and transported retrogradely to express a therapeutic agent in the cell bodies. In one example, short hairpin RNA (shRNA) against α-synuclein (which prevents the expression of α-synuclein in target neurons) is expressed from a retrograde form of a virus such as a retroAAV / rh10 vector and delivered to the vagal sensory fibers by injection into the wall of the stomach and / or intestine. The ultimate expression of shRNA in the vagal sensory neurons blocks the expression of endogenous α-synuclein in these neurons, thereby preventing the spread of toxic synuclein pathology from the pathological fibrils in the gastrointestinal tract, resulting in the widespread brain pathology observed in Parkinson's disease, as the propagation of pathological synuclein requires expression in neurons. In another example, a retroAAV / rh10 vector within the vagal sensory fibers delivered via the gastrointestinal tract expresses an antibody against α-synuclein to prevent the spread.

[0084] In one embodiment, a method of delivering a gene to nerve fibers that control organ function is provided, the method comprising delivering a gene therapy mediator to a target organ region innervated by a regulatory nerve. In one embodiment, the nerve fibers are selected from the group consisting of the vagus nerve, the cardio-pulmonary nerves, the thoracic splanchnic nerves, the lumbar splanchnic nerves, the sacral splanchnic nerves, and the pelvic splanchnic nerves. In one embodiment, the organs are selected from the group consisting of the stomach, intestine, pancreas, liver, lung, heart, adrenal gland, kidney, gonad, bladder, anal sphincter, and urethral sphincter. In one embodiment, the gene therapy mediator is a viral mediator selected from the group consisting of adeno-associated virus, lentivirus, adenovirus, and herpes simplex virus. In one embodiment, the gene therapy mediator is a modified viral mediator selected for retrograde transport in the central nervous system, including retrograde forms of adeno-associated virus and lentivirus, and canine adenovirus.

[0085] In one embodiment, a method of modulating organ function to ameliorate a disease, the method comprising delivering a gene capable of modulating neuronal activity to a nerve controlling the organ by injecting a gene therapy mediator into the organ for uptake and retrograde transport in neurons. In one embodiment, the nerve fibers are selected from the group consisting of the vagus nerve, the cardio-pulmonary nerves, the thoracic splanchnic nerves, the lumbar splanchnic nerves, the sacral splanchnic nerves, and the pelvic splanchnic nerves. In one embodiment, the organs are selected from the group consisting of the stomach, intestine, pancreas, liver, lung, heart, adrenal gland, kidney, gonad, bladder, anal sphincter, and urethral sphincter. In one embodiment, the viral mediator is selected from the group consisting of adeno-associated virus, lentivirus, adenovirus, and herpes simplex virus. In one embodiment, a modified viral mediator selected for retrograde transport in the central nervous system is employed, including retrograde forms of adeno-associated virus and lentivirus, and canine adenovirus. In one embodiment, the delivered gene modulates the activity of the nerve controlling the organ in response to an exogenous agent or stimulus. In one embodiment, the delivered gene encodes one or more light-sensitive ion channels (optogenetics), chemically-responsive ion channels (chemogenetics), ultrasound-sensitive ion channels (sonogenetics), magnetic field-responsive ion channels (magnetogenetics), and designer receptors exclusively activated by designer drugs (DREADD).

[0086] In one embodiment, a method of controlling food intake is provided, by injecting a vector into a visceral organ to deliver a viral vector to vagus nerve fibers to reduce food intake. In one embodiment, the organ is the stomach, duodenum or small intestine. In one embodiment, the delivered gene modulates the activity of the nerves of the organ in response to exogenous agents or stimuli. In one embodiment, the delivered gene encodes one or more photosensitive ion channels (optogenetics), chemically responsive ion channels (chemogenetics), ultrasound-sensitive ion channels (sonogenetics), magnetic field-responsive ion channels (magnetogenetics), and designer receptors exclusively activated by designer drugs (DREADD).

[0087] In one embodiment, a method of preventing cough is provided by delivering a viral vector to nerve fibers of the lung by inhalation of the viral vector. In one embodiment, the delivered gene modulates the activity of the nerves of the organ in response to exogenous agents or stimuli. In one embodiment, the gene encodes one or more proteins, including one or more photosensitive ion channels (optogenetics), chemically responsive ion channels (chemogenetics), ultrasound-sensitive ion channels (sonogenetics), magnetic field-responsive ion channels (magnetogenetics), and designer receptors exclusively activated by designer drugs (DREADD).

[0088] In one embodiment, a viral vector for improving retrograde delivery and uptake into neurons that control visceral organs is provided. In one embodiment, the vector includes an adeno-associated viral vector that carries a capsid including, for example, a capsid mixture from AAV retro and AAVrh10.

[0089] In one embodiment, a method of preventing the spread of toxic proteins from the gastrointestinal tract to the brain is provided by delivering a viral vector expressing a gene capable of blocking the spread of toxic proteins to the vagus nerve. In one embodiment, the viral vector is retrogradely taken up from the gastrointestinal system. In one embodiment, the targeted toxic proteins are selected from the group consisting of α-synuclein, τ protein, β-amyloid protein, or huntingtin protein. In one embodiment, the genes expressed from the vagus nerve to prevent the spread of toxic proteins are selected from the group consisting of short hairpin RNA (shRNA), microRNA (miRNA), CrispR / Cas9, antibodies, single-chain antibodies, or intracellular antibodies.

[0090] In one embodiment, a method of preventing, suppressing, or treating cough in a mammal is provided. The method comprises indirectly delivering (e.g., by injection) a composition comprising a viral vector to parasympathetic nerve fibers innervating the lungs of the mammal, the viral vector comprising a gene encoding a protein whose activity is inhibited by administering an exogenous agent or delivering energy; and exposing the mammal to an amount of an agent or energy effective to prevent, suppress, or treat cough in the mammal. In one embodiment, the composition is administered by inhalation. In one embodiment, the mammal is a human. In one embodiment, the mammal has idiopathic cough or intractable cough. In one embodiment, the mammal has chronic obstructive pulmonary disease (COPD) or gastric reflux. In one embodiment, the viral vector is an adeno-associated virus, lentivirus, adenovirus, or herpes simplex virus vector. In one embodiment, the virus is a retrograde form of adeno-associated virus (AAV), lentivirus, or canine adenovirus. In one embodiment, the virus is modified for retrograde transport. In one embodiment, the AAV has a capsid comprising proteins from more than one AAV serotype. In one embodiment, the capsid proteins are AAV2 and AAVrh10. In one embodiment, the capsid proteins are AAV5 and AAVrh10. In one embodiment, the capsid proteins are AAV2 and AAV5. In one embodiment, the gene encodes a light-sensitive ion channel (optogenetics), a chemically responsive ion channel (chemogenetics), an ultrasound-sensitive ion channel (sonogenetics), a magnetic field-responsive ion channel (magnetogenetics), or a designer receptor exclusively activated by designer drugs (DREADD).

[0091] In one embodiment, a method of preventing, suppressing, or treating coughing is provided, comprising delivering a composition comprising an effective amount of a composition to nerve fibers of the mammalian lung, the composition comprising a viral vector comprising a gene. In one embodiment, the expression of the gene inhibits neuronal activity. In one embodiment, the gene encodes a DREADD or some other chemogenetic channel, GAD for producing GABA to inhibit neurons, or an siRNA for blocking an excitatory protein or channel. In one embodiment, the composition is administered by inhalation or injection, such as directly to the vagus nerve or the nodose ganglion. In one embodiment, the gene modulates the activity of the nerves of the organ in response to an exogenous agent or stimulus. In one embodiment, the mammal is a human. In one embodiment, the mammal has idiopathic cough or intractable cough. In one embodiment, the mammal has COPD or gastric reflux. In one embodiment, the viral vector is an adeno-associated virus, lentivirus, adenovirus, or herpes simplex virus vector. In one embodiment, the virus is a retrograde form of adeno-associated virus, lentivirus, or canine adenovirus. In one embodiment, the virus is modified for retrograde transport. In one embodiment, the gene encodes a light-sensitive ion channel (optogenetics), a chemically-responsive ion channel (chemogenetics), an ultrasound-sensitive ion channel (sonogenetics), a magnetic field-responsive ion channel (magnetogenetics), or a designer receptor exclusively activated by designer drugs (DREADD). In one embodiment, the viral vector is an rAAV comprising a chimeric adeno-associated virus capsid, the chimeric adeno-associated virus capsid comprising two or more different AAV capsid serotypes. In one embodiment, the biological vector transduces vagal afferents. In one embodiment, one of the AAV serotypes comprises AAV2. In one embodiment, one of the AAV serotypes comprises AAVrh10.

[0092] In one embodiment, a method is provided for delivering a gene to a nerve fiber to control the function of a visceral organ in a mammal. In one embodiment, the visceral organ does not include the brain or muscle. The method includes delivering a composition comprising a viral vector to one or more regions of a mammalian organ innervated by a regulatory nerve, the viral vector including a gene encoding a protein whose activity is inhibited by administering an exogenous agent or delivering energy; and exposing the mammal to an effective amount of the agent or energy. In one embodiment, the nerve fiber is the vagus nerve, cardio-pulmonary nerve, thoracic visceral nerve, lumbar visceral nerve, sacral visceral nerve, or pelvic visceral nerve. In one embodiment, the mammalian organ to be controlled is the stomach, intestine, pancreas, liver, lung, heart, adrenal gland, kidney, gonad, bladder, anal sphincter, or urethral sphincter. In one embodiment, the composition is administered to the stomach, intestine, pancreas, liver, lung, heart, adrenal gland, kidney, gonad, bladder, anal sphincter, or urethral sphincter, or a blood vessel, duct, or other cavity. In one embodiment, the viral vector is an adeno-associated virus, lentivirus, adenovirus, or herpes simplex virus vector. In one embodiment, the viral vector provides retrograde transport in neurons. In one embodiment, the virus is modified to provide retrograde transport in the central nervous system. In one embodiment, the virus is a retrograde form of an adeno-associated virus, lentivirus, or canine adenovirus. In one embodiment, the gene modulates the activity of the nerve innervating the organ in response to an exogenous agent or stimulus. In one embodiment, the gene encodes a light-sensitive ion channel (optogenetics), a chemically-responsive ion channel (chemogenetics), an ultrasound-sensitive ion channel (sonogenetics), a magnetic-field-responsive ion channel (magnetogenetics), or a designer receptor exclusively activated by designer drugs (DREADD). In one embodiment, the gene encodes a light-sensitive channel protein (e.g., TREK-1), nicotinic acetylcholine receptor, gramicidin A, voltage-gated potassium channel, ionotropic glutamate channel, Na V1.5, KCNQ1, KCNA, MEC-4, DEG / ENaC / ASIC ion channels or mechanosensitive ion channels (MscL), such as TRPV4. In one embodiment, the composition is delivered to the vagus nerve. In one embodiment, the amount administered permits control of food intake in a mammal. In one embodiment, the visceral organ is the stomach, duodenum, or small intestine. In one embodiment, the gene encodes a gene product capable of blocking the spread of a toxic protein to the vagus nerve of a mammal. In one embodiment, the viral vector is retrogradely taken up from the gastrointestinal system. In one embodiment, the toxic protein includes alpha-synuclein, tau protein, beta-amyloid protein, or huntingtin protein. In one embodiment, the gene encodes a short hairpin RNA (shRNA), microRNA (miRNA), CrispR / Cas9, antibody, single-chain antibody, or intracellular antibody. In one embodiment, the amount administered prevents or inhibits the spread of a toxic protein from the gastrointestinal tract to the brain in a mammal. In one embodiment, the mammal is a human. In one embodiment, the viral vector is an adeno-associated virus, lentivirus, adenovirus, or herpes simplex virus vector. In one embodiment, the virus is modified to provide retrograde transport in the central nervous system. In one embodiment, the virus is the retrograde form of an adeno-associated virus, lentivirus, or canine adenovirus. In one embodiment, one of the AAV serotypes includes AAV2. In one embodiment, one of the AAV serotypes includes AAVrh10. In one embodiment, the viral vector is an rAAV comprising a chimeric adeno-associated virus capsid, the chimeric adeno-associated virus capsid comprising two or more different AAV capsid serotypes. In one embodiment, the amount administered prevents, inhibits, or treats a disease in the visceral organ in the mammal.

[0093] In one embodiment, there is provided an rAAV comprising a capsid formed of capsid proteins from two or more different AAV serotypes (chimeric AAV capsid). In one embodiment, one of the serotypes comprises AAV2. In one embodiment, one of the serotypes comprises AAVrh10. In one embodiment, one capsid serotype comprises AAV2 and the other comprises AAVrh10. In one embodiment, one capsid serotype comprises AAV2 and the other comprises AAV1, AAV3, AAV5, AAV8 or AAV9. In one embodiment, one capsid serotype comprises AAVrh10 and the other comprises AAV1, AAV2, AAV3, AAV5, AAV8 or AAV9. In one embodiment, one capsid serotype comprises AAV5 and the other comprises AAV1, AAV2, AAV3, AAV8 or AAV9. In one embodiment, one capsid serotype comprises AAV9 and the other comprises AAV1, AAV2, AAV3, AAV5 or AAV8. In one embodiment, one capsid serotype provides retrograde delivery. In one embodiment, the rAAV encodes a therapeutic gene product, a prophylactic gene product or an exogenously activatable protein.

[0094] The present invention will be further described by the following non-limiting examples.

[0095] Example 1

[0096] The figure shows a method for controlling feeding behavior by modulating gene therapy. Obesity is one of the most common public health problems. Over the past 25 years, more than 700 million people worldwide have been obese (BMI > 30 kg / m 2 ), and the prevalence has doubled (GBD obesity collaborators, Health Effects of Overweight and Obesity in 195 Countries Over 25 years, NEJM 377:13, 2017). In the United States, more than 78 million people with a BMI > 30 are obese, associated with a shortened lifespan and an increased risk of multiple diseases, including diabetes, cardiovascular disease, and cancer. Severe obesity (> 40 kg / m 2) represents a rapidly growing population with a strong unmet need. In 2010, there were approximately 15 million in the United States, and this is expected to increase to 25 million by 2025 (Finkelstein et al., Obesity and Severe Obesity Forecasts through 2030, Am J Prev Med 42:563, 2012). In randomized trials, losing 5%-15% of overweight body weight can reduce the risk factors for cardiovascular and other diseases (Office of Surgeon General, Call to Action to Prevent and Decrease Overweight and Obesity, 2001).

[0097] Bariatric surgery is currently the main option for severely obese patients. The most popular procedure is sleeve gastrectomy, which reduces the size of the stomach and induces early satiety. Although 15-25 million people meet the criteria for surgery (BMI > 40 or BMI 30-40 with severe weight-related health problems), only 228,000 bariatric surgeries were performed in the United States in 2017 ( https: / / asmbs.org / resources / estimate - of - bariatric - surgery - numbers ). More than 14% are revision surgeries. The post-operative treatment protocol requires 1-3 days of hospitalization, then only 7 days of liquid diet, 3 weeks of pureed food, and then a return to normal diet ( https: / / www.mayoclinic.org / tests - procedures / sleeve - gastrectomy / about / pac - 20385183 ). Due to malabsorption, daily multivitamin and calcium supplements are required, and monthly B12 injections. Bariatric surgery is associated with a complication rate of 13%-21%. Minimally invasive gene therapy methods will provide outpatient surgical options, require no post-operative management, and have a minimal risk of surgical complications.

[0098] Figure 1 Data showing delivery of AAV to the gastrointestinal tract to target the dorsal motor nucleus of the vagus nerve: mCherry signal in the dorsal motor nucleus of the vagus nerve. Different AAV vectors were injected into the gastric body (1×10 12 p / ml) along with fluorescently labeled cholera toxin subunit B (CTB-594) as a positive control. On the day of surgery, these mice also received an intraperitoneal injection of the retrograde tracer Fluoro-Gold to label gastrointestinal afferent and efferent neuron cell bodies. Four weeks after surgery, the nodose ganglia and brains were harvested for histological analysis of the mCherry red fluorescent protein, which was expressed as a cassette under the chicken β-actin promoter by the AAV vector (AAV.CBA.mCherry).

[0099] Figure 2 shows delivery of AAV to the gastrointestinal tract to target the dorsal motor nucleus of the vagus nerve: mCherry signal in the nodose ganglia. Different AAV vectors were injected into the gastric body (1×10 12p / ml), and fluorescently labeled cholera toxin subunit B (CTB-594) as a positive control. On the day of surgery, these mice also received an intraperitoneal injection of the retrograde tracer Fluoro-Gold to label the cell bodies of gastrointestinal afferent and efferent neurons. Four weeks after surgery, the nodose ganglia and brains were harvested for histological analysis of the mCherry red fluorescent protein, which was expressed as a cassette under the chicken β-actin promoter by an AAV vector (AAV.CBA.mCherry).

[0100] Figure 3 are the feeding behavior data of fasted mice at 1 mg / kg CNO, and FIG. 4 is the feeding behavior data of normally fed mice at 1 mg / kg CNO.

[0101] A route for delivering viral vectors to control organ function is shown in Figure 5 . For example, a composition having rAAV encoding hM3Dq (see, e.g., SEQ ID No. 10 or 11 in FIG. 16, which is modified to DREADD hM3Dq) can be delivered, e.g., to the greater curvature of the stomach, by endoscopy or laparoscopy, which, in one embodiment, allows delivery of the virus to the vagal afferent nerves. In some embodiments, administration can allow delivery to the vagal efferent nerves or motor neurons. In one embodiment, the composition is delivered to visceral organs, including but not limited to the heart, liver, lung, adrenal gland, thyroid gland, pancreas, intestine, kidney, bladder, or spleen.

[0102] In one embodiment, the chimeric AAV capsid comprises a ratio of one AAV serotype (e.g., AAV2) to another AAV serotype (e.g., AAVrh10) capsid of 0.1:1, 0.5:1, 1:1, 1:3, 1:4, 1:5, 1:15, 1:20, 1:50, 1:100, 1:500.

[0103] Example 2

[0104] The cough reflex is generally important for expelling potential obstructions or contaminants from the lung airways. Intractable cough is typically treated by attempting to address the underlying cause of the cough, such as gastric reflux, asthma, or chronic inflammation caused by chronic obstructive pulmonary disease or malignancy. However, for many people, chronic cough is the primary problem, and there is no clear ongoing irritant or stimulant that can be addressed. This is similar to intractable pain, which may be caused by an underlying pathology that needs to be reversed, but often the pain itself needs to be addressed because there is no abnormality that can be reversed. For patients with intractable cough without reversible pathology, there are few treatment options. Narcotics can be used to attempt to suppress the cough, but these have significant morbidity, especially with long-term use.

[0105] To inhibit coughing in an animal model, retroAAV2 / rh10 expressing the inhibitory DREADD hM4Di (1×10 12 p / ml) (see, e.g., SEQ ID NO:12 of M4 in Figure 16, which is modified to DREADD hM4Di) was nebulized and sprayed into the trachea and upper airways of guinea pigs. Guinea pigs were used because they exhibit a strong cough reflex, while rats and mice do not have an effective cough reflex. Six weeks after exposure, histological evaluation using immunostaining confirmed the expression of inhibitory opsin in a subset of neurons in the nodose ganglion that provide sensation to the trachea and upper airways. Then a second group of guinea pigs was divided into two subgroups. One subgroup was again administered the same retroAAV2 / rh10. The hM4Di nebulized agent was sprayed into the upper airways, while the second subgroup received retroAAV2 / rh10 expressing mCherry as a negative control. Six weeks later, the animals were placed in an enclosed plexiglass chamber. The chamber contained a pressure monitor for measuring changes in the chamber air pressure due to coughing and also contained a microphone that could capture coughing sounds. These allowed continuous monitoring of the frequency, total number, and intensity of coughing, and the monitors were time-locked to confirm that a true cough occurred only when both the sound change and the pressure change matched. To induce coughing, the chamber was then filled with nebulized 2M citric acid at a rate of 5L / min, which was sufficiently diluted so that it would not cause permanent harm to the animals but would irritate the airways and cause coughing. Then both subgroups were exposed to the acidic gas, and the number, frequency, and intensity of coughing in the two groups were evaluated within 10 minutes to confirm that there were no differences between the two groups at baseline. A third group of untreated guinea pigs was exposed to the citric acid chamber to confirm that the presence of the retrograde agent and the nebulized spray did not affect baseline coughing compared to untested animals. In the second segment, then all subgroups were administered 1mg / kg CNO for 30 minutes before being placed in the acidic gas chamber. Then the number, frequency, and intensity of coughing were quantified and compared between and within subgroups under both the agent condition and the CNO condition. This confirmed that guinea pigs receiving AAV with inhibitory DREADDs coughed less when exposed to the acidic gas compared to before administration of CNO and compared to the mCherry and untested control groups.

[0106] All publications, patents, and patent applications are incorporated herein by reference. Although the invention has been described in the foregoing specification in connection with its specific embodiments and numerous details have been set forth for purposes of illustration, it will be apparent to those skilled in the art that the invention admits of additional embodiments and that certain details herein may be subject to considerable variation without departing from the basic principles of the invention.

Claims

1. A recombinant adeno-associated virus (rAAV), wherein the rAAV comprises a capsid formed by a capsid protein comprising SEQ ID NO:5 and a capsid protein comprising SEQ ID NO:

7.

2. The rAAV according to claim 1, wherein the rAAV provides retrograde delivery.

3. The rAAV according to claim 1, wherein the rAAV encodes a therapeutic gene product, a prophylactic gene product, or an exogenously controllable protein.

4. The rAAV according to claim 3, wherein the rAAV encodes a light-sensitive ion channel, a chemically responsive ion channel, an ultrasound-sensitive ion channel, a magnetic field-responsive ion channel, or a DREADD.

5. The rAAV according to claim 3, wherein the rAAV encodes a channel protein or a DREADD that is inhibited by an exogenously administered agent.

6. The rAAV according to claim 5, wherein the rAAV encodes hD3q.

7. The rAAV according to claim 2, wherein the rAAV provides retrograde transport in the central nervous system.

8. The rAAV according to claim 3, wherein the rAAV encodes a gene product that regulates the activity of parasympathetic fibers innervating visceral organs in response to the administration of an agent.

9. The rAAV according to claim 3, wherein the rAAV encodes a light-sensitive channel protein, a nicotinic acetylcholine receptor, gramicidin A, a voltage-gated potassium channel, an ionotropic glutamate receptor, alpha-hemolysin, or a mechanically sensitive channel.

10. The rAAV according to claim 3, wherein the rAAV encodes a gene product that blocks the spread of a toxic protein to the vagus nerve of a mammal.

11. The rAAV according to claim 2, wherein the rAAV is retrogradely taken up from the gastrointestinal system.

12. The rAAV according to claim 10, wherein the toxic protein comprises alpha-synuclein, tau protein, beta-amyloid protein, or huntingtin protein.

13. The rAAV according to claim 3, wherein the rAAV encodes a gene product that comprises or encodes a short hairpin RNA (shRNA), a microRNA (miRNA), CrispR / Cas9, or an antibody.

14. The rAAV according to claim 13, wherein the antibody is a single-chain antibody.

15. The rAAV according to claim 13, wherein the antibody is an intracellular antibody.

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