Compositions and methods of delivering a therapeutic across the blood-brain barrier via the PL16 receptor

Abstract

The present invention provides engineered AAV vectors, and methods of using vectors, comprising capsid proteins that comprise a sequence that binds to the PL16 protein. In some embodiments, it includes a membrane protein expressed in several cell populations and tissues in the body, including brain microvascular endothelial cells in humans. By the present invention, it was discovered that engineered novel AAV vectors comprising a capsid protein having a sequence that allows the vector to bind to P116 may have an increased ability to provide a gene therapy which crosses the blood brain barrier.

Description

[0001] COMPOSITIONS AND METHODS OF DELIVERING A THERAPEUTIC ACROSS THE BLOOD-BRAIN BARRIER VIA THE PL 16 RECEPTOR

[0002] Field of the Invention

[0003] The disclosure relates to compositions and methods for the preparation, use, and / or formulation of vectors with increased crossing of the blood brain barrier (BBB).

[0004] Background

[0005] Treatments for central nervous system diseases are frequently limited by the ability to cross the blood-brain barrier (BBB), which almost entirely limits the passage of many therapeutic drugs into the central nervous system (CNS).

[0006] Gene therapies, for example based on the adeno-associated virus (AAV) vector, have been proposed as a treatment for a variety of genetic diseases. However, widely studied AAV serotypes have been shown to exhibit limited efficiency in treating CNS diseases. The emergence of AAV directed evolution technology has been proposed as a solution to the challenges presented by the BBB.

[0007] Nevertheless, attempts at providing compositions with improved ability to cross the blood brain barrier have been met with limited success and there remains a need for improved methods of producing and delivering active agents of interest to a target cell or tissue, e.g., a CNS cell or tissue.

[0008] Summary

[0009] The present invention provides engineered AAV vectors, and methods of using vectors, comprising capsid proteins that comprise a sequence that binds to the peptidase inhibitor 16 (PH6) protein, a membrane protein expressed in several cell populations and tissues in the body, including brain microvascular endothelial cells in humans. By the present invention, it was discovered that engineered novel AAV vectors comprising a capsid protein having a sequence that allows the vector to bind to P116 may have an increased ability to provide a gene therapy which crosses the blood brain barrier.

[0010] Without being limited by a mechanism of action, this increased ability to cross the blood brain barrier may be provided by PI16 mediated transcytosis (i.e. receptor mediated transport of extracellular cargo across the cytoplasm of a cell to a different plasma membrane surface). Moreover, and advantageously, because transcytosis is different from AAV transduction and involves different interactions, off-target transduction of cells expressing PI16 may be limited, resulting in robust expression of a transgcnc in the CNS with limited off-target effects.

[0011] Aspects of the invention provide an engineered AAV vector comprising an engineered capsid protein comprising an amino acid sequence that allows the capsid protein to bind to peptidase inhibitor 16 (PI16) protein. The engineered AAV may further comprise a nucleic acid encoding a transgene, with the nucleic acid encapsidated by the engineered capsid protein.

[0012] The engineered AAV vector may be derived from an AAV9 vector. Advantageously, the AAV vector may be modified from the AAV9 vector to exhibit tissue specificity and to comprise the sequence that binds to the PI16 protein.

[0013] For example, the amino acid sequence that allows the capsid protein to bind to PI16 comprises a substitution at the AA588 relative to an AAV9 vector and peptide insertion between AA588 and AA589 relative to an AAV9. The amino acid sequence that allows capsid protein to bind PI16 may include other substitutions or insertions in variable regions of the capsid.

[0014] Advantageously, the AAV vector may be characterized by at least one of an increased specificity and / or increased transduction efficiency in the central nervous system (CNS) tissue. The AAV capsid protein may be characterized by at least one of an increased specificity and / or increased transduction efficiency in the brain.

[0015] Further advantageously, the AAV capsid protein is characterized by delivery of the transgene across the blood brain barrier (BBB).

[0016] The transgene may be a therapeutic transgene for the treatment of a disorder affecting the central nervous system. The PI16 protein may be human PI16.

[0017] For example, the transgene may be AADC, ARSA, ASP, CLN2, CLN6, FXN, GAD, GAL, GBA1, GCL, GDNF, NAGLU, NGF, NEP / IDE, NPY, NRIA, NTN, SCN1A, SNC2A, SNC8A, SCN1B, SCN2B, SGSH, SMN, SMN1, STXB1

[0018] Notably, when provided to a cell expressing PI16 as a surface protein, binding of the capsid protein to the PI16 protein may mediate delivery of the transgene across the BBB, for example via transcytosis.

[0019] Aspects of the invention also provide methods of delivering a transgene across the BBB. Methods of the invention may comprise providing a subject (e.g. a human subject) an engineered AAV vector comprising an engineered capsid protein comprising an amino acid sequence that allows the AAV vector to bind to the PI16 protein and a transgene encapsidated by the capsid protein. Binding the AAV vector to the PT 16 protein in cells expressing the PI16 protein thereby mediates delivery of the transgcnc across the BBB.

[0020] Methods of the invention utilize AAV vectors of the invention. Accordingly, in methods of the invention, the engineered AAV vector may be derived from an AAV9 vector. The amino acid sequence that allows the capsid protein to bind to PI16 may comprise a substitution at the AA588 relative to an AAV9 vector and peptide insertion between AA588 and AA589 relative to an AAV9. The capsid protein that binds to PI16 may comprise substitutions or insertions in other residues of the capsid, for example between AA452 and AA458 relative to AAV9.

[0021] Advantageously, the AAV vector may exhibit increased specificity for and / or increased transduction efficiency in the CNS tissue. The AAV capsid protein may exhibit increased specificity for and / or increased transduction efficiency in the brain.

[0022] Without being bound to a cell-type expression profile, the cells expressing the PI16 protein may be microvascular endothelial cells. Advantageously, the AAV capsid protein may exhibit specificity for microvascular endothelial cells. Binding of the capsid protein to the PI16 protein may mediate delivery of the transgene across the BBB via transcytosis.

[0023] Aspects of the invention further provide uses and compositions for use comprising engineered AAV capsid proteins of the invention for delivery of a transgene across the BBB. Accordingly, aspects of the invention provide methods and uses for treatment of CNS disorders that comprise administration to a subject in need of AAV vectors of the invention.

[0024] Delivery of AAVs of the invention may be provided by intravenous injection. Advantageously, an AAV capsid of the invention may allow for blood brain barrier penetration following intravenous administration. In some embodiments, the AAV capsid variant allows for blood brain barrier penetration following intravenous administration, focused ultrasound (FUS), e.g., coupled with the intravenous administration of microbubbles (FUS-MB), or MRI-guided FUS coupled with intravenous administration.

[0025] In some embodiments, routes for administration include administration into the CSF, for example, via an intracerebroventricular (ICV), intrathecal cisternal, intra cisterna magna, or intrathecal lumbar route. Particular embodiments result in delivery to neurons and glial cells of the brain. Other routes of delivery to the CNS / brain include, but are not limited to intracranial administration, lateral cerebroventricular administration, intranasal administration, endovascular administration, and intraparenchymal administration. Administration to a subject may be by intraparenchymal administration. Tn some embodiments, the intraparenchymal administration is to tissue of the central nervous system.

[0026] Accordingly, intravenous delivery of AAVs to the subject results in transduction of CNS tissues. For example, CNS tissue may include tissue of the brain, including thalamus, cortex, putamen, lateral ventricles, medulla, the pons, the amygdala, the motor cortex, caudate, hypothalamus, striatum, ventral midbrain, neocortex, basal ganglia, hippocampus, substantia, nigra, cerebrum, cerebellum, and from brainstem and spinal cord.

[0027] Brief Description of the Drawings

[0028] FIG. 1 is a table of vectors (test / control) screened against a protein library.

[0029] FIG. 2 shows the spotting pattern for slides used in the protein library confirmation screen.

[0030] FIG. 3A-D show slides of the protein library screen following treatment with the AAV capsids of the invention and AAV9 control to fixed confirmation slides.

[0031] FIG. 4A-D show slides of the protein library screen following treatment with the AAV capsids of the invention and AAV9 control in the absence of fixation.

[0032] FIG. 5 is a summary table of overall specific interactions from the confirmation screen with weak intensity and above.

[0033] FIG. 6 shows data from a protein capture experiment, demonstrating binding of AAV capsids of the invention to bead-bound human PI16.

[0034] FIG. 7 shows data from the recovery of AAV capsids of the invention and human PI16 on SDS-PAGE gel after binding and subsequent elution.

[0035] FIG. 8 shows data from binding of AAV capsids of the invention to human PI 16 in platebased ELISA.

[0036] FIG. 9 shows data from cell-based overexpression experiments, with increased transduction and expression by AAV capsids of the invention of cell culture after overexpression of PI16.

[0037] Detailed Description

[0038] The present invention provides engineered AAV vectors, and methods of using vectors, comprising capsid proteins that comprise a sequence that binds to the PI16 protein, a protein which, without being bound to cell-type expression profiles, may be expressed on the membrane of brain microvascular of endothelial cells in humans.

[0039] By the present invention, it was discovered that engineered novel AAV vectors comprising a capsid protein having a sequence that allows the vector to bind to PI16 may have an increased ability to provide a gene therapy which crosses the blood brain barrier. peptidase inhibitor 16 (PI16)

[0040] Without being bound to a cell-type expression profile, PI16 is a protein that may be expressed on the membrane of brain microvascular endothelial cells in humans. PI16 is mainly expressed in fibroblasts, however PI 16 is found throughout the body in several cell populations and tissues. The protein may also be expressed in neural progenitor cells and cells in the pleura of humans.

[0041] By the present invention, it was found that PI16 may mediate transcytosis across the blood brain barrier.

[0042] Transgene delivery

[0043] In some embodiments, an individual is treated by a method comprising administering a therapeutically effective amount of one or more compositions encompassed herein, including any viral particle herein, to the individual. The composition may increase the level of a heterologous transgene in the individual, including in cells of the individual. In some embodiments, the composition administered to the individual restores the level of the transgene to a level found in a control individual. In some embodiments, the compositions restore cognitive abilities in the individual. In some embodiments, the compositions reduce the number and / or severity of seizures in the individual. In some embodiments, the compositions restore motor functions in the individual. In some embodiments, the compositions restore psychiatric and behavioral functions in the individual.

[0044] Transgene delivery strategies may result in gene replacements or gene supplementation, used interchangeably herein. Specifically, gene replacement specifically refers to introduction of exogenous nucleic acids to host cells to restore gene expression of a mutated or deleted gene. Gene supplementation refers to the introduction of exogenous nucleic acids to host cells to increase a specific gene's expression in the context of mutations or deletions that result in reduced gene expression from host DNA.

[0045] The transgene may be in cis with two inverted terminal repeats (ITRs) flanking the transgene. Due to the limited packaging capacity of the rAAV (~5kB), in some cases, the transgene may be split between two AAV vectors, the first with 3’ splice donor and the second with a 5’ splice acceptor. Upon co-infection of a cell, concatemers form, which are spliced together to express a full-length transgene.

[0046] Effective dosages of the viral particles to be administered to a subject will depend upon the mode of administration, the disease or condition to be treated, the individual subject's condition, the particular virus or viral vector, and the nucleic acid to be delivered, and can be determined in a routine manner. Examples of effective doses for achieving therapeutic effects include virus titers of at least about 105, 106, 107, 108, 109, IO10, 1011, 1012, 1013, 1014, 1015transducing units or more.

[0047] In some embodiments, viral particles are administered directly to the CNS, e.g., the brain or the spinal cord. Direct administration can result in high specificity of transduction of CNS cells, e.g., wherein at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the transduced cells are CNS cells. However, it is understood that transduction may be as low as 10% while maintaining therapeutic efficacy, for example when crossing the blood brain barrier. Any method known in the art to administer vectors directly to the CNS can be used. The vector may be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (corpus striatum, cerebrum including the occipital, temporal, parietal and frontal lobes, cortex, basal ganglia, hippocampus, and amygdala), limbic system, neocortex, corpus striatum, cerebrum, and inferior colliculus. The vector may also be administered to different regions of the eye such as the retina, cornea, or optic nerve. The vector may be delivered into the cerebrospinal fluid (e.g., by lumbar puncture) for more disperse administration of the vector.

[0048] The delivery vector may be administered to the desired region(s) of the CNS by any route known in the ail, including but not limited to, intrathecal, intracerebral, intra-cistema magna, intraventricular, intranasal, intra- aural, intra-ocular (e.g., intra-vitreous, sub-retinal, anterior chamber) and peri ocular (e.g., sub-Tcnon's region) delivery or any combination thereof.

[0049] Typically, the viral vector will be administered in a liquid formulation by direct injection to the desired region or compartment in the CNS. In some embodiments, the vector can be delivered via a reservoir and / or pump. In other embodiments, the vector may be provided by topical application to the desired region or by intra-nasal administration of an aerosol formulation. Administration to the eye or into the ear, may be by topical application of liquid droplets. As a further alternative, the vector may be administered as a solid, slow-release formulation.

[0050] In some embodiments, one can inject the AAV particles directly into the brain tissues. In some embodiments, one can deliver the particles into cerebrospinal fluid (CSF), such as by injection into the ventricle or lumbar intrathecal space. In some embodiments, one can deliver the particles systemically, such as by injection into a blood vessel, and then let the AAV particles cross the blood brain barrier (BBB).

[0051] For example, the present invention may provide engineered AAV capsid proteins comprising a capsid sequence that binds to the PI16 protein.

[0052] By the present invention, it was discovered that engineered novel AAV vectors comprising capsid protein having an insert that allows the capsid protein to bind to PI16 may have an increased ability to provide a gene therapy which crosses the blood brain barrier.

[0053] As a result, and advantageously, in particular embodiments, one or more AAV particles of the disclosure have the ability to cross the BBB. In embodiments wherein any AAV particle is considered to have very weak or no ability to cross BBB, one can inject the AAV particles directly into the brain tissues, such as by intrap arenchymal injection.

[0054] In general, methods disclosed herein comprise administering a therapeutic rAAV composition by systemic administration. In some instances, methods comprise administering a therapeutic rAAV composition by intraperitoneal injection. In some instances, methods comprise administering a therapeutic rAAV composition by intravenous (“IV”) administration. It is conceivable that one may also administer therapeutic rAAV compositions disclosed herein by other routes, such as subcutaneous injection, intramuscular injection, intradermal injection, transdermal injection percutaneous administration, intranasal administration, intralymphatic injection, rectal administration, intragastric administration, intraocular administration, intracerebroventricular administration, intrathecally, or any other suitable parenteral administration. Routes, dosage, time points, and duration of administrating therapeutics may be adjusted. In some embodiments, administration of therapeutics is prior to, or after, onset of either, or both, acute and chronic symptoms of the disease or condition.

[0055] The term “CNS” or “central nervous system” means a tissue selected from brain, including thalamus, cortex, putamen, lateral ventricles, medulla, the pons, the amygdala, the motor cortex, caudate, hypothalamus, striatum, ventral midbrain, neocortex, basal ganglia, hippocampus, substantia, nigra, cerebrum, cerebellum, and from brainstem and spinal cord. The brain includes a variety of cortical and subcortical areas, including the frontal, temporal, occipital, and parietal lobes.

[0056] The term “systemic delivery” is defined as a route of administration of medication or other substance into a circulatory system so that the entire body is affected. Administration can take place via enteral administration (absorption of the drug through the gastrointestinal tract) or parenteral administration (generally injection, infusion, or implantation). “Circulatory system” includes both blood or cerebrospinal fluid circulatory systems. Examples of systemic administration for the CNS include intraarterial, intravenous or intrathecal injection. Other examples include administration to the cerebrospinal fluid at any location, in the spine (i.e., but not limited to lumbar) or brain (i.e., but not limited to cistema magna). The terms “systemic administration” and “systemic delivery” are used interchangeably.

[0057] In some embodiments, routes for administration include administration into the CSF, for example, via an intracerebroventricular (ICV), intrathecal cisternal, intra cistema magna, or intrathecal lumbar route. Particular embodiments result in delivery to neurons and glial cells of the brain. Other routes of delivery to the CNS / brain include, but are not limited to intracranial administration, lateral cerebroventricular administration, intranasal administration, endovascular administration, and intraparenchymal administration.

[0058] An effective dose and dosage of pharmaceutical compositions to prevent or treat the disease or condition disclosed herein is defined by an observed beneficial response related to the disease or condition, or symptom of the disease or condition. Beneficial response comprises preventing, alleviating, arresting, or curing the disease or condition, or symptom of the disease or condition. In some embodiments, the beneficial response may be measured by detecting a measurable improvement in the presence, level, or activity, of biomarkers, transcriptomic risk profile, or intestinal microbiome in the subject. An “improvement,” as used herein refers to shift in the presence, level, or activity towards a presence, level, or activity observed in normal individuals (e.g. individuals who do not suffer from the disease or condition). The dosage amount and / or route of administration may be changed, or an additional agent may be administered to the subject, along with the therapeutic rAAV composition. In some embodiments, as a patient is started on a regimen of a therapeutic rAAV composition, the patient is also weaned off (e.g., step-wise decrease in dose) a second treatment regimen.

[0059] In some cases, a dose of the pharmaceutical composition may comprise a concentration of infectious particles of at least or about 107, 108, 109, IO10, 1011, 1012, 1013, 1014, 1015, 1016, or 1017. In some cases, the concentration of infectious particles is 2xl07, 2xl08, 2xl09, 2xlO10, 2xlOn, 2xl012, 2xl013, 2xl014, 2xl015, 2xl016, 2xl017. In some cases, the concentration of the infectious particles is 3xl07, 3xl08, 3xl09, 3xlO10, 3xlOn, 3xl012, 3xl013, 3xl014, 3xl015, 3xl016, or 3xl017. In some cases, the concentration of the infectious particles is 4xl07, 4xl08, 4xl09, 4xlO10, 4xlOn, 4xl012, 4xl013, 4xl014, 4xl015, 4xl016, or 4xl017. In some cases, the concentration of the infectious particles is 5xl07, 5xlO8, 5xl09, 5xlO10, 5xlOn, 5xl012, 5xl013, 5xl014, 5xl015, 5xl016, or 5xl017. In some cases, the concentration of the infectious particles is 6xl07, 6xl08, 6xl09, 6xlO10, 6xlOn, 6xl012, 6xl013, 6xl014, 6xl015, 6xl016, or 6xl017. In some cases, the concentration of the infectious particles is 7xl07, 7xl08, 7xl09, 7xlO10, 7xlOn, 7xl012, 7xl013, 7xl014, 7xl015, 7xl016, or 7xl017. In some cases, the concentration of the infectious particles is 8xl07, 8xlO8, 8xl09, 8xlO10, 8xlOn, 8xl012, 8xlO13, 8xl014, 8xl015, 8xl016, or 8xl017. In some cases, the concentration of the infectious particles is 9xl07, 9xl08, 9xl09, 9xlO10, 9xlOn, 9xl012, 9xl013, 9xl014, 9xl015, 9xl016, or 9xl017. It is understood that the dose of the pharmaceutical compositions may comprise a concentration of infectious particles that is between the above concentrations.

[0060] Disclosed herein, in some embodiments are formulations of pharmaceutically-acceptable excipients and carrier solutions suitable for delivery of the rAAV compositions described herein, as well as suitable dosing and treatment regimens for using the particular compositions described herein in a variety of treatment regimens. In some embodiments, the amount of therapeutic gene expression product in each therapeutically-useful composition may be prepared in such a way that a suitable dosage will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, as well as other pharmacological considerations will he contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable. In some instances, the rAAV compositions are suitably formulated pharmaceutical compositions disclosed herein, to be delivered either intraocularly, intravitreally, parenterally, subcutaneously, intravenously, intracerebroventricularly, intramuscularly, intrathecally, intraperitoneally, by nasal inhalation, or by direct injection to one or more cells, tissues, or organs by direct injection.

[0061] In some embodiments, the pharmaceutical forms of the AAV-based viral compositions suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and / or vegetable oils. Proper fluidity may be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0062] In some cases, for administration of an injectable aqueous solution, for example, the solution may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, and the general safety and purity standards as required by FDA Office of Biologies standards.

[0063] Disclosed herein are sterile injectable solutions comprising the rAAV compositions disclosed herein, which are prepared by incorporating the rAAV compositions disclosed herein in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions arc prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. Injectable solutions may be advantageous for systemic administration, for example by intravenous administration.

[0064] Also provided herein are formulations in a neutral or salt form. Pharmaceutically- acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug-release capsules, and the like.

[0065] Suitable dose and dosage administrated to a subject is determined by factors including, but not limited to, the particular therapeutic rAAV composition, disease condition and its severity, the identity (e.g., weight, sex, age) of the subject in need of treatment, and can be determined according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, the condition being treated, and the subject or host being treated.

[0066] The amount of AAV compositions and time of administration of such compositions will be within the purview of the skilled artisan having benefit of the present teachings. It is likely, however, that the administration of therapeutically-effective amounts of the disclosed compositions may be achieved by a single administration, for example, a single injection of sufficient numbers of infectious particles to provide therapeutic benefit to the patient undergoing such treatment. This is made possible, at least in part, by the fact that certain target cells (e.g., neurons) do not divide, obviating the need for multiple or chronic dosing.

[0067] For example, the number of infectious particles administered to a mammal may be on the order of about 107, 108, 109, IO10, 1011, 1012, 1013, 1014, or in between these orders or higher even higher, infectious particles / ml given either as a single dose or divided into two or more administrations as may be required to achieve therapy of the particular disease or disorder being treated. In fact, in certain embodiments, it may be desirable to administer two or more different AAV vector compositions, either alone, or in combination with one or more other therapeutic drugs, to achieve the desired effects of a particular therapy regimen. In various embodiments, the daily and unit dosages are altered depending on a number of variables including, but not limited to, the activity of the therapeutic rAAV composition used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

[0068] The effective dosage ranges may be adjusted based on subject’s response to the treatment. Some routes of administration will require higher concentrations of effective amount of therapeutics than other routes.

[0069] In certain embodiments, the daily dosage range and / or the unit dosage amount varies within this range depending upon the dosage form employed and the route of administration utilized.

[0070] Viral Vectors

[0071] Certain embodiments of the disclosure concern methods of producing viral particles. In some embodiments, the method comprises providing to a cell in vitro, (a) a template comprising (i) a nucleic acid encoding for a gene product, and (ii) packaging signal sequences sufficient for the encapsidation of an AAV template into virus particles (e.g., one or more (e.g., two) terminal repeats, such as AAV terminal repeats), and (b) AAV sequences sufficient for replication and encapsidation of the template into viral particles (e.g., the AAV rep and AAV cap sequences encoding an AAV capsid). The template and AAV replication and capsid sequences are provided under conditions such that recombinant virus particles comprising the template packaged within the capsid are produced in the cell. The method can further comprise the step of collecting the virus particles from the cell. Virus particles may be collected from the medium and / or by lysing the cells.

[0072] Recombinant adeno-associated virus (rAAV) mediated gene delivery leverages the AAV mechanism of viral transduction for nuclear expression of an episomal heterologous nucleic acid (e.g., a transgene, therapeutic nucleic acid). For example, upon delivery to a host in vivo environment, a rAAV may (1) bind or attach to cellular surface receptors on the target cell, (2) endocytose, (3) traffic to the nucleus, (4) uncoat the virus to release the encapsidated heterologous nucleic acid, (5) convert of the heterologous nucleic acid from single- stranded to double-stranded DNA as a template for transcription in the nucleus, and (6) transcribe of the episomal heterologous nucleic acid in the nucleus of the host cell. rAAVs engineered to have an increased specificity (binding to cellular surface receptors on the target cell), transduction efficiency (the effectiveness of a virus, engineered or naturally occurring, at delivering its DNA component to a host cell), and transgene expression (transcription of the episomal heterologous nucleic acid in the host cell) are desirable for gene therapy applications.

[0073] An rAAV comprises an AAV capsid that can be engineered to encapsidate a heterologous nucleic acid (e.g., therapeutic nucleic acid, gene editing machinery). The AAV capsid is made up of three AAV capsid protein monomers, VP1, VP2, and VP3. Sixty copies of these three VP proteins interact in an approximately 1:1:10 ratio to form the viral capsid. VP1 covers the whole of VP2 protein in addition to a -137 amino acid N-terminal region (VPlu), VP2 covers the whole of VP3 in addition to -65 amino acid N-terminal region (VP1 / 2 common region). The three capsid proteins share a conserved amino acid sequence of VP3, which in some cases is the region beginning at amino acid position 138 (e.g., AA139-736).

[0074] While not wishing to be bound by theory, it is understood that a parent AAV capsid sequence comprises a VP1 region. In certain embodiments, a parent AAV capsid sequence comprises a VP1, VP2 and / or VP3 region, or any combination thereof. A parent VP1 sequence may be considered synonymous with a parent AAV capsid sequence.

[0075] The AAV VP3 structure contains highly conserved regions that are common to all serotypes, a core eight-stranded P-barrel motif (PB-0I) and a small a-helix (aA). The loop regions inserted between the P-strands consist of the distinctive HI loop between P-strands H and I, the DE loop between p-strands D and E, and nine variable regions (VRs), which arc typically surface exposed on the capsid structure. These VRs, such as VR-VIII, which contains AA588 in AAV9, can be associated with specific functional roles in the AAV life cycle, including receptor binding, transduction, and antigenic specificity.

[0076] Disclosed herein are AAV capsids comprising AAV capsid proteins with a substitution at the AA588 and peptide insertion between AA588 and AA589 that confer a desired tropism characterized by a higher efficiency and specificity for transduction in specific cell-types, including, for example, cells within the CNS or brain cell types (e.g., brain endothelial cells, neurons, astrocytes). In particular, the AAV capsid proteins disclosed herein enable rAAV- mediated transduction of a heterologous nucleic acid (e.g., transgene) in the CNS of a subject. The AAV capsids of the present disclosure, or the AAV capsid proteins, may be formulated as a pharmaceutical composition. In addition, the AAV capsids or the AAV capsid proteins can be isolated and purified to be used for a variety of applications. Disclosed herein are recombinant AAV (rAAV) capsids which comprise AAV capsid proteins that are engineered with a modified capsid protein (e.g., VP1, VP2, VP3). In some embodiments, the rAAV capsid proteins of the present disclosure are generated using the methods disclosed herein. In some embodiments, the AAV capsids are used in the methods of delivering a therapeutic nucleic acid (e.g., a transgene) to a subject. In some instances, the rAAV capsids have desired AAV tropisms rendering them particularly suitable for certain therapeutic applications, e.g., the treatment of a disease or disorder in a subject such as those disclosed herein.

[0077] The rAAV capsid proteins are engineered for optimized transduction and transgene expression in the CNS, for example the brain, of a subject upon systemic administration of the rAAV to the subject. The rAAV capsid proteins are engineered to have tropisms that eliminate the need for intracranial injection, while also achieving widespread and efficient transduction of an encapsidated transgene. In particular, the tropisms comprise at least one of an increased specificity and efficiency (e.g., of viral transduction) in the CNS of a subject, as compared to a reference AAV.

[0078] The engineered AAV capsid proteins described herein have, in some cases, a peptide insertion and amino acid substitution that is heterologous to the parental AAV capsid protein at the amino acid positions 587 and 590 in AAV9. In some embodiments, the amino acids flanking the peptide insertion do not originate from the parental AAV capsid protein. The amino acids flanking the insertion may share sequence identity with the amino acids at the same position within the parental serotype or equivalent amino acid position as the substitution and peptide insertion in alternative AAV serotypes or engineered variant capsid proteins.

[0079] Also disclosed herein are rAAVs with engineered capsid proteins that are optimized for targeting specific organ or tissue within a subject. In a non-limiting example, the rAAVs of the present embodiment have increased specificity, transduction, and transgene expression in the CNS.

[0080] Seven amino acids comprise the peptide insertion sequence (7-mer, respectively) that is inserted or substituted within VR-VIII in the parental AAV capsid protein. Aspects provided herein provide amino acid insertions comprising seven amino acid polymer (7-mer) inserted between AA588-589 and may additionally include a substitution of one or two amino acids at amino acid positions flanking the 7-mer sequence (e.g., AA587-588 and / or AA589-590) to produce an eleven amino acid polymer (11-mer) at the 588 loop of a parental AAV capsid protein.

[0081] Peptide insertion sequences of the disclosure include sequences that have been modified in any way and for any reason, for example, to: (1) reduce susceptibility to proteolysis, (2) alter binding affinities, and (3) confer or modify other physicochemical or functional properties. For example, single or multiple amino acid substitutions (e.g., equivalent, conservative or nonconservative substitutions, deletions, or additions) may be made in a sequence.

[0082] An AAV vector can comprise a viral genome comprising a nucleic acid sequence encoding the recombinant AAV (rAAV) capsid protein described herein. The viral genome can comprise a Replication (Rep) gene encoding a Rep protein, and Capsid (Cap) gene encoding an AAP protein in the first open reading frame (ORF1) or a Cap protein in the second open reading frame (ORF2). The Rep protein is selected from Rep78, Rep68, Rep52, and Rep40. In some instances, the Cap gene is modified encoding a modified AAV capsid protein described herein. A wild-type Cap gene encodes three proteins, VP1, VP2, and VP3. In some cases, VP1 is modified. In some cases, VP2 is modified. In some cases, VP3 is modified. In some cases, all three VP1- VP3 are modified. The AAV vector can comprise nucleic acids encoding wild-type Rep78, Rep68, Rep52, Rep40 and AAP proteins.

[0083] In some instances, the 5' ITR and the 3' ITR are derived from an AAV2 serotype. In some instances, the 5' ITR and the 3' ITR are derived from an AAV5 serotype. In some instances, the 5' ITR and the 3' ITR are derived from an AAV9 serotype. In some instances, the 5’ ITR and the 3’ ITR each originate from different serotypes, e.g. 5’ ITR from serotype AAV2 and 3’ ITR from AAV5. In some instances, the 5’ ITR and / or the 3’ ITR originate from another natural serotype or have been engineered for improved transduction or transgene expression efficiency.

[0084] A conservative amino acid substitution refers to the substitution of an amino acid in an insertion sequence with a functionally similar amino acid having similar properties, e.g., size, charge, hydrophobicity, hydrophilicity, and / or aromaticity.

[0085] In some embodiments, methods of increasing transduction of an encoded gene in a target in vivo environment comprise delivering a rAAV particle described herein, the rAAV engineered to have an increased transduction enrichment in a target in vivo environment (e.g., tissue or cell type). In some instances, the increased transduction enrichment comprises a 1-fold, 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50- fold or 100-fold increase, or more, relative to a reference AAV. In some instances, the increased transduction enrichment is at least 2-fold. In some instances, the increased transduction enrichment is at least 10-fold. In some instances, the increased transduction enrichment is at least 20-fold.

[0086] Methods of delivering a heterologous nucleic acid to a target in vivo environment are also provided comprising delivering the rAAV particle described herein that has been engineered to have an increased expression or specificity in an in vivo environment (e.g., tissue or cell type), as compared to a reference AAV. Methods, in some cases, comprise detecting whether a rAAV possesses more specificity for an in vivo environment, including measuring a level of gene expression product expressed from the vector encapsidated by the rAAV in a tissue sample obtained from the in vivo environment in a subject.

[0087] In some instances, the reference AAV has a serotype selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, or variants thereof.

[0088] Provided herein are methods of delivering a heterologous nucleic acid to a target in vivo environment comprising delivering a composition to the target in vivo environment selected from a CNS in a subject, the composition comprising a rAAV particle with a rAAV capsid protein, the rAAV capsid protein encapsidating a viral vector encoding a heterologous nucleic acid (e.g., therapeutic nucleic acid). In some embodiments, the rAAV particle encapsidating the heterologous nucleic acid comprises a rAAV capsid protein engineered with an increased transduction enrichment when measured in the CNS of the subject, even when administered to the subject systemically.

[0089] Methods may comprise delivering a rAAV particle comprising an rAAV capsid protein with increased transduction and transgene expression efficiency when measured in the CNS in the subject. In some embodiments, delivery is systemic. Alternatively, delivery is direct (e.g., into the affected area of the CNS).

[0090] Promoter / Enhancers

[0091] A variety of promoter / enhancer elements may be used depending on the level and tissuespecific expression desired. The promoter / enhancer may be constitutive or inducible, depending on the pattern of expression desired. The promoter / enhancer may be native or foreign and can be a natural or a synthetic sequence. By foreign, it is intended that the transcriptional initiation region is not found in the wild-type host into which the transcriptional initiation region is introduced.

[0092] Promoter / enhancer elements can be native to the target cell or subject to be treated and / or native to the heterologous nucleic acid sequence. The promoter / enhancer element is generally chosen so that it will function in the target cell(s) of interest. In representative embodiments, the promoter / enhancer element is a mammalian promoter / enhancer element. The promoter / enhance element may be constitutive or inducible.

[0093] Promoters are DNA regions that initiate gene transcription by controlling the binding of RNA polymerase to the vector DNA to begin the process toward expression of the encoded protein. Promoters control the binding of RNA polymerase to DNA. RNA polymerase transcribes DNA to mRNA which is ultimately translated into a functional protein. Thus, the promoter region controls when and where in the organism the gene of interest is expressed. Exemplary promoters include CMV, CBh, human synapsin I, EFla, SV40, PGK1, Ubc, human beta actin, and CAG. In preferred embodiments, the vector comprises a promoter selected from a CAG synthetic promoter, a CBh synthetic promoter, and a human synapsin I promoter. See Miyazaki, J; Takaki, S; Araki, K; Tashiro, F; Tominaga, A; Takatsu, K; Yamamura, K (Jul 15, 1989). "Expression vector system based on the chicken beta-actin promoter directs efficient production of interleukin-5". Gene. 79 (2): 269-77; Grey et al., Optimizing Promoters for Recombinant Adeno-Associated Virus-Mediated Gene Expression in the Peripheral and Central Nervous System Using Self-Complementary Vectors, Hum Gene Ther. 201 1 Sep; 22(9): 1143- 1153; Glover ct al., Adcnoviral-mcdiatcd, High-Level, Cell-Specific Transgcnc Expression: A SYN1-WPRE Cassette Mediates Increased Transgene Expression With No Loss of Neuron Specificity, Mol Ther. 2002 May; 5(5 Pt 1 ):509- 16; the content of each of which is incorporated herein by reference.

[0094] In some instances, the vector may comprise a promoter and / or enhancer, for example, a constitutive promoter or an inducible or tissue / cell specific promoter. As a non-limiting example, the promoter may be CMV promoter, a CMV-P-Actin-intron-P-Globin hybrid promoter (CAG), CBA promoter, FRDA or FXN promoter, UBC promoter, GUSB promoter, NSE promoter, Synapsin promoter, MeCP2 promoter, GFAP promoter, Hl promoter, U6 promoter, NFL promoter, NFH promoter, SCN8A promoter, or PGK promoter. As a non-limiting example, promoters can be tissue- specific expression elements that include, but are not limited to, human elongation factor la-subunit (EFla), immediate-early cytomegalovirus (CMV), chicken P-actin (CBA) and its derivative CAG, the P glucuronidase (GUSB), and ubiquitin C (UBC). The vector may include a tissue-specific expression elements for neurons such as, but not limited to, neuronspecific enolase (NSE), platelet-derived growth factor (PDGF), platelet-derived growth factor Bchain (PDGF-P), the synapsin (Syn), the methyl-CpG binding protein 2 (MeCP2),Ca2+ / calmodulin-dependent protein kinase II (CaMKII), metabotropic glutamate receptor (mGluR2), NFL, NFH, np32, PPE, Enk and EAAT2 promoters. The vector may comprise a tissue-specific expression element for astrocytes such as, but not limited to, the glial fibrillary acidic protein (GFAP) and EAAT2 promoters. The vector may comprise tissue-specific expression elements for oligodendrocytes such as, but not limited to, the myelin basic protein (MBP) promoter.

[0095] Various regulatory elements may be included in vectors of the invention including posttranscriptional regulatory elements (PREs) such as those derived from hepatitis B virus (HPRE), woodchuck hepatitis virus (WPRE), human heat shock protein 70 mRNA (Hsp70), the vascular endothelial growth factor (SP163), the tripartite leader sequence of human adenovirus mRNA linked with a major late promoter enhancer (TM), or the first intron of human cytomegalovirus immediate early gene (Intron A). Posttranscriptional regulatory elements can help enhance gene expression when included in expression vectors such as those described herein. Particular PREs may exhibit cell-specific and / or gene-specific regulatory enhancement and those factors arc considered when selecting a PRE.

[0096] Examples

[0097] AAV capsids of the invention were tested, in comparison to AAV9, for the ability to bind PI16 through multiple experimental approaches. Initially, AAV capsids of the invention were screened across a panel of cell-surface expressed proteins for binding. A confirmational screen against identified binding proteins was performed, identifying PI16 as a binding protein unique to AAV capsids of the invention in comparison to the AAV9 control capsid. Subsequently, binding of AAV capsids of the invention was measured through bead-based PI16 display and capture of AAV capsids of the invention, plate-based display of PI16 and enzyme-linked immunosorbent assay (ELISA) measurement of bound AAV capsid concentration, and assessment of novel AAV capsid transduction in cell culture, with and without overexpression of PI 16. Without being bound by a mechanism of action, PI 16 was identified as a potential receptor candidate for novel AAV capsids included in the invention.

[0098] Capsid Sequences of the Invention

[0099] Control Capsid (SEQ ID NO: 1):

[0100] MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD NARGLVLPGY

[0101] KYLGPGNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LKYNHADAEF

[0102] QERLKEDTSF GGNLGRAVFQ AKKRLLEPLG LVEEAAKTAP GKKRPVEQSP

[0103] QEPDSSAGIG KSGAQPAKKR LNFGQTGDTE SVPDPQPIGE PPAAPSGVGS

[0104] LTMASGGGAP VADNNEGADG VGSSSGNWHC DSQWLGDRVI TTSTRTWALP

[0105] TYNNHLYKQI SNSTSGGSSN DNAYFGYSTP WGYFDFNRFH CHFSPRDWQR

[0106] LINNNWGFRP KRLNFKLFNI QVKEVTDNNG VKTIANNLTS TVQVFTDSDY

[0107] QLPYVLGSAH EGCLPPFPAD VFMIPQYGYL TLNDGSQAVG RSSFYCLEYF

[0108] PSQMLRTGNN FQFSYEFENV PFHSSYAHSQ SLDRLMNPLI DQYLYYLSKT

[0109] INGSGQNQQT LKFSVAGPSN MAVQGRNYIP GPSYRQQRVS TTVTQNNNSE

[0110] FAWPGASSWA LNGRNSLMNP GPAMASHKEG EDRFFPLSGS LIFGKQGTGR

[0111] DNVDADKVMI TNEEEIKTTN PVATESYGQV ATNHQSAQAQ AQTGWVQNQG ILPGMVWQDR DVYLQGPIWA KIPHTDGNFH PSPLMGGFGM KHPPPQILIK NTPVPADPPT AFNKDKLNSF ITQYSTGQVS VEIEWELQKE NSKRWNPEIQ YTSNYYKSNN VEFAVNTEGV YSEPRPIGTR YLTRNL

[0112] Novel AAV Capsid 1 (SEQ ID NO: 2)

[0113] MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD NARGLVLPGY

[0114] KYLGPGNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LKYNHADAEF

[0115] QERLKEDTSF GGNLGRAVFQ AKKRLLEPLG LVEEAAKTAP GKKRPVEQSP

[0116] QEPDSSAGIG KSGAQPAKKR LNFGQTGDTE SVPDPQPIGE PPAAPSGVGS

[0117] LTMASGGGAP VADNNEGADG VGSSSGNWHC DSQWLGDRVI TTSTRTWALP

[0118] TYNNHLYKQI SNSTSGGSSN DNAYFGYSTP WGYFDFNRFH CHFSPRDWQR

[0119] LINNNWGFRP KRLNFKLFNI QVKEVTDNNG VKTIANNLTS TVQVFTDSDY

[0120] QLPYVLGSAH EGCLPPFPAD VFMIPQYGYL TLNDGSQAVG RSSFYCLEYF

[0121] PSQMLRTGNN FQFSYEFENV PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT

[0122] INGSGQNQQT LKFSVAGPSN MAVQGRNYIP GPSYRQQRVS TTVTQNNNSE

[0123] FAWPGASSWA LNGRNSLMNP GPAMASHKEG EDRFFPLSGS LIFGKQGTGR

[0124] DNVDADKVMI TNEEEIKTTN PVATESYGQV ATNHQSATRN GEIFIAQAQT

[0125] GWVQNQGILP GMVWQDRDVY LQGPIWAKIP HTDGNFHPSP LMGGFGMKHP

[0126] PPQILIKNTP VPADPPTAFN KDKLNSFITQ YSTGQVSVEI EWELQKENSK RWNPEIQYTS NYYKSNNVEF AVNTEGVYSE PRPIGTRYLT RNL

[0127] Novel AAV Capsid 2 (SEQ ID NO: 3)

[0128] MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD NARGLVLPGY

[0129] KYLGPGNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LKYNHADAEF

[0130] QERLKEDTSF GGNLGRAVFQ AKKRLLEPLG LVEEAAKTAP GKKRPVEQSP

[0131] QEPDSSAGIG KSGAQPAKKR LNFGQTGDTE SVPDPQPIGE PPAAPSGVGS

[0132] LTMASGGGAP VADNNEGADG VGSSSGNWHC DSQWLGDRVI TTSTRTWALP

[0133] TYNNHLYKQI SNSTSGGSSN DNAYFGYSTP WGYFDFNRFH CHFSPRDWQR

[0134] LINNNWGFRP KRLNFKLFNI QVKEVTDNNG VKTIANNLTS TVQVFTDSDY

[0135] QLPYVLGSAH EGCLPPFPAD VFMIPQYGYL TLNDGSQAVG RSSFYCLEYF

[0136] PSQMLRTGNN FQFSYEFENV PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT INGSGQNQQT LKFSVAGPSN MAVQGRNYIP GPSYRQQRVS TTVTQNNNSE

[0137] FAWPGASSWA LNGRNSLMNP GPAMASHKEG EDRFFPLSGS LIFGKQGTGR

[0138] DNVDADKVMI TNEEEIKTTN PVATESYGQV ATNHQSATRN GEVFIAQAQT

[0139] GWVQNQGILP GMVWQDRDVY LQGPIWAKIP HTDGNFHPSP LMGGFGMKHP

[0140] PPQILIKNTP VPADPPTAFN KDKLNSFITQ YSTGQVSVEI EWELQKENSK RWNPEIQYTS NYYKSNNVEF AVNTEGVYSE PRPIGTRYLT RNL

[0141] Novel AAV Capsid 3 (SEQ ID NO: 4)

[0142] MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD NARGLVLPGY KYLGPGNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LKYNHADAEF QERLKEDTSF GGNLGRAVFQ AKKRLLEPLG LVEEAAKTAP GKKRPVEQSP

[0143] QEPDSSAGIG KSGAQPAKKR LNFGQTGDTE SVPDPQPIGE PPAAPSGVGS

[0144] LTMASGGGAP VADNNEGADG VGSSSGNWHC DSQWLGDRVI TTSTRTWALP

[0145] TYNNHLYKQI SNSTSGGSSN DNAYFGYSTP WGYFDFNRFH CHFSPRDWQR LINNNWGFRP KRLNFKLFNI QVKEVTDNNG VKTIANNLTS TVQVFTDSDY

[0146] QLPYVLGSAH EGCLPPFPAD VFMIPQYGYL TLNDGSQAVG RSSFYCLEYF

[0147] PSQMLRTGNN FQFSYEFENV PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT

[0148] INGTGQNQQT LKFSVAGPSN MAVQGRNYIP GPSYRQQRVS TTVTQNNNSE

[0149] FAWPGASSWA LNGRNSLMNP GPAMASHKEG EDRFFPLSGS LIFGKQGTGR

[0150] DNVDADKVMI TNEEEIKTTN PVATESYGQV ATNHQSATRN GEVFIAQAQT

[0151] GWVQNQGILP GMVWQDRDVY LQGPIWAKIP HTDGNFHPSP LMGGFGMKHP

[0152] PPQILIKNTP VPADPPTAFN KDKLNSFITQ YSTGQVSVEI EWELQKENSK RWNPEIQYTS

[0153] NYYKSNNVEF AVNTEGVYSE PRPIGTRYLT RNL

[0154] Novel AAV Capsid 4 (SEQ ID NO: 5)

[0155] MAADGYLPDW LEDNLSEGIR EWWALKPGAP QPKANQQHQD NARGLVLPGY

[0156] KYLGPGNGLD KGEPVNAADA AALEHDKAYD QQLKAGDNPY LKYNHADAEF

[0157] QERLKEDTSF GGNLGRAVFQ AKKRLLEPLG LVEEAAKTAP GKKRPVEQSP

[0158] QEPDSSAGIG KSGAQPAKKR LNFGQTGDTE SVPDPQPIGE PPAAPSGVGS

[0159] LTMASGGGAP VADNNEGADG VGSSSGNWHC DSQWLGDRVI TTSTRTWALP

[0160] TYNNHLYKQI SNSTSGGSSN DNAYFGYSTP WGYFDFNRFH CHFSPRDWQR LINNNWGFRP KRLNFKLFNI QVKEVTDNNG VKTIANNLTS TVQVFTDSDY

[0161] QLPYVLGSAH EGCLPPFPAD VFMIPQYGYL TLNDGSQAVG RSSFYCLEYF

[0162] PSQMLRTGNN FQFSYEFENV PFHSSYAHSQ SLDRLMNPLI DQYLYYLSRT

[0163] INGPVSSGKQNQQT LKFSVAGPSN MAVQGRNYIP GPSYRQQRVS TTVTQNNNSE

[0164] FAWPGASSWA LNGRNSLMNP GPAMASHKEG EDRFFPLSGS LIFGKQGTGR

[0165] DNVDADKVMI TNEEEIKTTN PVATESYGQV ATNHQSATRN GEVFIAQAQT

[0166] GWVQNQGILP GMVWQDRDVY LQGPIWAKIP HTDGNFHPSP LMGGFGMKHP

[0167] PPQILIKNTP VPADPPTAFN KDKLNSFITQ YSTGQVSVEI EWELQKENSK RWNPEIQYTS

[0168] NYYKSNNVEF AVNTEGVYSE PRPIGTRYLT RNL

[0169] Cell surface protein screen

[0170] A library of 3 x 104particles / cell of novel AAV capsids included in the invention was screened for binding against fixed HEK293 cells expressing duplicate 6105 human plasma membrane, secreted, or cell surface tethered human secreted proteins, 400 human heterodimers, and 218 non-human primate (Macaca fascicularis) plasma membrane and tethered secreted proteins arrayed across cell microarray slide sets (n=2 per slide set).

[0171] Particles used are described in the table below:

[0172] All transfection efficiencies exceeded the minimum threshold. A biotinylated anti-AAV9 detection antibody (1:500, as sold by Thermofisher; Cat # 7103332500, Lot # 220317-01) followed by AlexaFluor647 streptavidin detection reagent was used.

[0173] 19 library interactions (duplicate spots) were identified by analyzing fluorescence (AF647 and ZsGreenl) on ImageQuant. A range of intensities (signal to background) were identified from very weak to weak. Confirmation screen of cell surface expression binding

[0174] Confirmation screens of cell surface expression binding were conducted using vectors encoding all 19 interactors identified in the library screen. A vector encoding KIAA0319L (isoform 1), in duplicate, plus a control vector encoding EGFR in quadruplicate, were spotted on new slides and used to reverse transfect HEK293 cells, as before. All transfection efficiencies exceeded the minimum threshold.

[0175] FIG. 1 is a table of vectors (test / control) screened against a protein library.

[0176] Transfected HEK293 cells were treated after fixation or in the absence of fixation with 3 x 104particles / cell of the novel AAV capsid included in the invention , 3 x 104particles / cell of a control article, or no test molecule (biotinylated Anti-AAV9 and streptavidin or streptavidin only; negative controls) (n=2 slides per treatment for the fixed cell screen and n=l slide per treatment in the absence of fixation). Novel AAV capsids included in the invention showed significant specific interactions with AAV receptor KIAA0319L (custom synthesis) and IL3, both after fixation and in the absence of fixation. These interactions were also observed with control article, confirming prior interactions expected for the AAV9 serotype.

[0177] No interactions were identified for the novel AAV capsid when screened against the non-human primate (Macaca fascicitlari.s) library.

[0178] FIG. 2 shows the spotting pattern for slides.

[0179] Slides were analyzed as above. Interactions were categorized as specific or non-specific (i.e. the interaction also observed with the negative controls).

[0180] FIG. 3A-D show slides following treatment with the AAV capsids of the invention and AAV9 control to fixed confirmation slides.

[0181] FIG. 4A-D show slides following treatment with the AAV capsids of the invention and AAV9 control in the absence of fixation.

[0182] Interactions of weak intensity or above, specific to the test AAV, were identified.

[0183] FIG. 5 is a summary table of overall specific interactions from the confirmation screen with weak intensity and above. Assessment of PH 6 protein binding to AAV capsids via bead-based capture

[0184] Magnetic agarose beads enabling peptide specific capture (Pierce™ Anti-DYKDDDDK Magnetic Agarose) were used to bind purified DYKDDDDK-tagged human PI16 (produced in mammalian cells) and washed to remove any unbound protein. Subsequently, AAV capsids were applied to the PI16 labeled beads. After an incubation period, beads were washed to remove unbound viral particles, and a low-pH solution was applied to elute PI16-capsid complexes. The elution was neutralized and prepared for analysis. ddPCR of the AAV-packaged transgene was used to quantitatively compare bound capsids to total capsids applied to the PI16 labeled beads. Additional AAV capsid sequences tested in this experiment are shown below.

[0185] FIG. 6 shows data from a protein capture experiment, demonstrating binding of AAV capsids of the invention to bead-bound human PI16. The recovery of AAV capsid, calculated through ddPCR targeting WPRE element on the vector genome, is plotted as a percentage of the concentration applied to PI16 bound beads. Higher values indicate binding of the AAV capsid to PI16, while lower values indicate a lack of binding interaction to PI16. Engineered capsids of the invention bind PI16 (% Recovery 39-60%), while parental serotype AAV9 does not bind (0.3% Recovery). Bars represent N = 1 sample replicates per condition.

[0186] FIG. 7 shows the recovery of AAV capsids of the invention, demonstrated by SDS-Page of PI16 and AAV capsid proteins eluted from a DYKDDDDK-tag (SEQ ID NO: 6) magnetic bead capture. Column A indicates the applied capsid, and column B is the elution following binding to PI16. Darker bands indicate a higher concentration of protein. Bands corresponding to human PI 16 and AAV VP3 are indicated. Minimal AAV9 is recovered during elution of human PI 16, while a large proportion of AAV capsids of the invention are eluted together with human PI16. These data indicate a binding interaction between AAV capsids of the invention and human PI16.

[0187] Assessment of PI16 binding to AAV capsids via Plate-Based methods

[0188] A colorimetric ELISA (Enzyme-Linked Immunosorbent Assay) specific to the FLAG peptide (sequence DYKDDDDK, SEQ ID NO: 6) tag was used to detect capsid-specific receptor binding using FLAG-tagged human PI16 protein. Tagged human PI16 protein was applied to FLAG-tagged Millipore Sigma Anti-Flag M2 plates. The plate was then washed and blocking buffer was added to prevent any nonspecific binding. After the plate was re-washed, samples containing AAV capsids at linear range doses were then incubated in the PI16-bound plates to enable capsid binding. After incubation, the plates were washed with 0.1% PBST to remove unbound capsids. Subsequently, anti- AAV HRP antibody was applied to enable detection of PI16-bound capsids. The plate was washed again to remove residual antibody and then TMB reagent was applied and incubated without exposure to light. The TMB reaction was terminated by sulfuric acid addition, producing optical density (OD) that correlates with the concentration of bound AAV capsid. The OD of each sample was quantified on a plate reader to directly measure the quantity of PI16-bound AAV capsids.

[0189] Plotting the measured OD against the input capsid dose enables the determination of binding interaction between the AAV capsids of the invention and human PI16. Negative controls were included in the plate to determine the basal level of interaction with human PI 16 and the FLAG-tagged Millipore Sigma Anti-Flag M2 plates. The negative controls included: a detection antibody only sample to quantify non-specific detection antibody binding; a capture antibody and detection antibody sample to show non-specific binding of detection antibody and background of the capture antibody with detection antibody; and an analyte and detection antibody only sample to determine the non-specific binding of the analyte in the absence of a capture antibody. The goal of these negative controls is to set the expectation of background noise and to distinguish true signal from background. The average of the OD values from the negative controls were averaged and subtracted from the final OD values generated for samples being tested. Measured OD above the negative control for AAV capsids of the invention support a binding interaction with human PI16.

[0190] FIG. 8 shows ELISA-based measurements of AAV capsids of the invention binding plate-bound PI 16. Optical density values corresponding to the quantity of AAV capsid binding to human PI16 are plotted against the vector concentration of the samples. Dose-dependent increases of AAV capsid bound to human PI16 indicate a binding interaction for AAV capsids of the invention, while parental serotype AAV9 did not display binding to human PI16. N = 2-3 sample wells per concentration.

[0191] Assessment of PI16 binding to AAV capsids via cell-based overexpression methods

[0192] HEK293T cells were cultured in 96-well plate format and transfected with PI16 overexpression plasmid using a lipofectamine transfection reagent. Transfected cells were then infected with AAV capsids packaging GFP cargo under regulation of a ubiquitous promoter to enable positive identification of cell transduction. Separate cell culture wells were exposed to increasing concentrations of AAV capsid at multiplicity of infection ranging from 1 x 103vg / cell to 3 x 105vg / cell. AAV capsids that successfully transduced cells resulted in expression of green fluorescent protein (GFP) to enable the quantification of transduction. Negative controls included untreated cells, cells transfected with PI16 without any AAV infection, and untransfected cells infected with AAV capsid. After infection, cells were fixed with paraformaldehyde and imaged with a fluorescence microscope.

[0193] FIG. 9 shows cell transduction of select AAV capsids of the invention in comparison to AAV9 control, after infection at a range of doses with and without overexpression of PI16. AAV capsids of the invention were introduced in a dose-dependent manner to cell populations with and without PI16 overexpression. Additionally, parental serotype AAV9 was introduced to cells at the same doses. The overexpression of PI16 in cells increased transduction of R5.22 capsid by 20%, while AAV9 transduction was not influenced by PI16 overexpression. N = 3 cell culture wells per sample.

[0194] Summary

[0195] Novel AAV capsids included in the invention showed a specific interaction with PI16 across multiple orthogonal methods. Without being bound by a mechanism of action, PI16 was identified as a potential receptor candidate for novel AAV capsids included in the invention.

[0196] Modified sequences and percent identity

[0197] For sequences disclosed throughout this application, it is understood that nucleic acid molecules and peptides may comprise one or more substitutions, for example conservative substitutions, that allow sequences to continue to function. Accordingly, sequences may have at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence disclosed.

[0198] “Percent (%) sequence identity” with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that is identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that arc known, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software, or other software appropriate for nucleic acid sequences. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0199] In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a some % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program’s alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0200] As used herein, the terms “homologous,” “homology,” or “percent homology” when used herein to describe a nucleic acid sequence relative to a reference sequence, can be determined using the formula described by Karlin & Altschul 1990, modified as in Karlin & Altschul 1993. Such a formula is incorporated into the basic local alignment search tool (BLAST) programs of Altschul 1990. Percent homology of sequences can be determined using the most recent version of BLAST, as of the filing date of this application. Homologous sequences described herein include sequences having the same percentage identity as the indicated percentage homology. Sequences sharing a percentage identity are understood in the art to mean those sequences sharing the indicated percentage of same residues over the length of the reference sequence (e.g., the linker or leader sequences disclosed herein and in the sequence listing).

[0201] A “conservative substitution” refers to amino acid substitutions that do not significantly affect or alter binding characteristics of a particular protein. Generally, conservative substitutions are ones in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include a substitution found in one of the following groups: Group 1: Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3: Asparagine (Asn or N), Glutamine (Gin or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (He or I), Leucine (Leu or L), Methionine (Met or M), Valine (Vai or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trp or W). Additionally, or alternatively, amino acids can be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Vai, Leu, and He. Other conservative substitutions groups include sulfur-containing: Met and Cys; acidic: Asp, Glu, Asn, and Gin; small aliphatic, nonpolar, or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gin; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, He, Vai, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company. Variant proteins, peptides, polypeptides, and amino acid sequences of the present disclosure can, in certain embodiments, comprise one or more conservative substitutions relative to a reference amino acid sequence.

[0202] A “functional variant” refers to a polypeptide or polynucleotide that is structurally similar or substantially structurally similar to a parent or reference compound of this disclosure, but differs, in some contexts slightly, in composition (e.g., one base, atom, or functional group is different, added, or removed; or one or more amino acids are substituted, mutated, inserted, or deleted), such that the polypeptide or encoded polypeptide is capable of performing at least one function of the encoded parent polypeptide with at least 50% efficiency of activity of the parent polypeptide.

[0203] As used herein, a “functional portion” or “functional fragment” refers to a polypeptide or polynucleotide that comprises only a domain, motif, portion, or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% activity associated with the domain, portion, or fragment of the parent or reference compound.

[0204] In certain embodiments, a functional variant or functional portion or functional fragment each refers to a “signaling portion” of an effector molecule, effector domain, costimulatory molecule, or costimulatory domain. In other aspects, a functional variant or functional portion or functional fragment each refers to a linking function or a leader peptide function as disclosed herein. In certain aspects, a functional variant / portion / fragment refers to a linking function or a leader peptide function as described herein. In specific aspects, variant linkers and leader peptides are at least 60% as efficient, at least 70% as efficient, at least 80% as efficient, at least 90% as efficient, at least 95% as efficient, or at least 99% as efficient as the reference / parent polypeptides disclosed herein.

[0205] Incorporation by Reference

[0206] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

[0207] Equivalents

[0208] Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

Claims1. An engineered adeno-associated virus (AAV) vector comprising: an engineered capsid protein comprising an amino acid sequence that allows the capsid protein to bind to peptidase inhibitor 16 (PI16) protein.

2. The engineered AAV vector of claim 1, further comprising a nucleic acid encoding a transgene, wherein the nucleic acid encapsidated by the engineered capsid protein.

3. The engineered AAV vector of claim 2, wherein the engineered AAV vector is derived from an AAV9 vector.

4. The engineered AAV vector of claim 3, wherein the amino acid sequence that allows the capsid protein to bind to PT16 comprises a substitution at the AA588 relative to an AAV9 vector and peptide insertion between AA588 and AA589 relative to an AAV9.

5. The engineered AAV vector of claim 4, wherein the AAV vector is characterized by at least one of an increased specificity and / or increased transduction efficiency in the central nervous system (CNS) tissue.

6. The engineered AAV vector of claim 5, wherein the AAV capsid protein is characterized by at least one of an increased specificity and / or increased transduction efficiency in the brain.

7. The engineered AAV vector of claim 6, wherein the AAV capsid protein is characterized by delivery of the transgene across the blood brain barrier (BBB).

8. The engineered AAV vector of claim 7, wherein the transgene is a therapeutic transgene for the treatment of a disorder affecting the central nervous system.

9. The engineered AAV vector of claim 8, wherein the P116 protein is human P116.

10. The engineered AAV vector of claim 9, wherein when provided to a cell expressing PT16 as a surface protein, binding of the capsid protein to the PI16 protein mediates transcytosis of the transgene across the BBB.

11. A method of delivering a transgene across the BBB, the method comprising: providing a subject an engineered AAV vector comprising: an engineered capsid protein comprising an amino acid sequence that allows the AAV vector to bind to the PI 16 protein; and a transgene encapsidated by the capsid protein; binding the AAV vector to the PI16 protein in cells expressing the PI16 protein, thereby delivering the transgene across the BBB.

12. The method of claim 11, wherein the engineered AAV vector is derived from an AAV9 vector.

13. The method of claim 12, wherein the amino acid sequence that allows the capsid protein to bind to PI16 comprises a substitution at the AA588 relative to an AAV9 vector and peptide insertion between AA588 and AA589 relative to an AAV9.

14. The method of claim 13, wherein the AAV vector exhibits increased specificity and / or increased transduction efficiency in the CNS tissue.

15. The method claim 14, wherein the AAV capsid protein exhibits increased specificity and / or increased transduction efficiency in the brain.

16. The method of claim 15, wherein the transgene is a therapeutic transgene for the treatment of a disorder affecting the central nervous system.

17. The method of claim 15, wherein the subject is a human.

18. The method of claim of claim 16, wherein the cells expressing the PI 16 protein are microvascular endothelial cells.

19. The method of claim 18, wherein the AAV capsid protein exhibits specificity for microvascular endothelial cells.

20. The method of claim 19, wherein binding of the capsid protein to the PI16 protein mediates delivery of the transgene across the BBB via transcytosis.

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