Gene therapy for friedreich's ataxia

Abstract

Aspects of the invention provide an AAV product that delivers an FXN gene supplementation or replacement strategy throughout the human CNS and heart at levels of DNA biodistribution, RNA expression, and FXN protein levels expected to restore function in Friedrich's ataxia patients.

Description

[0001] Atorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent Application

[0002] GENE THERAPY FOR FRIEDREICH’ S ATAXIA

[0003] Field of the Invention

[0004] The invention generally relates to virus compositions for treating Friedrich’s Ataxia. Background

[0005] Friedreich’s Ataxia (FA) is a rare, hereditary, form of ataxia causing progressive decline in gait and limb control, fatigue, sensory loss, bone deformities, diabetes, and cardiomyopathy. FA is caused by an intronic triplicate repeat expansion that diminishes the expression of the frataxin (FXN) protein. FXN protein plays a critical role in cellular respiration, and in its absence, there is deficient iron-sulfur clustering in the mitochondria leading to impaired cell respiration, oxidative stress, and cell death. Owing to eventual cell death, the impact to functions controlled by non- regenerative cell types (neurons, cardiomyocytes) is most pronounced. The incidence of Friedreich’s Ataxia is approximately 1 in every 50,000 live births, affecting -5,000 patients in the US and -15,000 worldwide.

[0006] Symptoms generally present at puberty with progression of Friedrich’s ataxia varying from person to person. Generally, within 10 to 20 years after the appearance of the first symptoms, individuals with FA may need to use a wheelchair. In later stages of the disorder, people may become completely incapacitated. Patients frequently have a shorter than normal life expectancy, with the average life expectancy for FA patients being 37 years of age, with death typically precipitated by cardiomyopathy.

[0007] Currently, there are no approved disease modifying treatments for FA. Omaveloxolone is the standard of care, a small molecule that improves neurological function by activating the Nrf2 pathway to reduce inflammation. Patients may also take over-the-counter anti arrhythmic agents and anti-cardiac failure medications to treat heart disease. Supportive care includes physical therapy, exercise, and the use of walking aids or wheelchairs.

[0008] Recombinant adeno-associated viruses (rAAVs) are widely used as vectors for gene delivery in therapeutic applications because of their ability to transduce both dividing and nondividing cells, their long-term persistence as episomal DNA in infected cells, and their low immunogenicity. These characteristics make them appealing for therapeutic applications, such as gene therapy. However, systemic delivery of existing AAV serotypes (e.g., intravenous, intrathecal, intraarterial, intracranial, intracisterna magna, intraventricular, intracerebroventricular, Atorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent Application intracardiac, or subcutaneous) shows limited transduction of certain cell types and organs, and non-specific, overlapping tropisms in others. This leads to several complications in gene therapy applications, including but not limited to off-target effects due to transduction of unimpacted organs and cell types (for example, the liver), and the necessity for a larger viral dosage to achieve sufficient therapeutic levels in the tissue or organ of interest.

[0009] Summary

[0010] Compositions and methods of the invention use recombinant adeno-associated viruses (rAAV) to deliver a viral vector comprising a frataxin (FXN) gene encoding a functional frataxin (FXN) protein.

[0011] Earlier and ongoing work in the field of gene therapy attempted to deliver the FXN cargo to the heart of FA patients using wild-type AAV9 serotypes or engineered variants for cardiacspecific targeting (rhlO). Treating cardiac symptoms of FA alone did not impact the aspects of disease most important to the patient’s quality of life, driven by impaired function in the central nervous system (CNS) including the brain, spinal cord, and retina. Additional ongoing efforts have been attempted to deliver the FXN cargo to the brain with direct injection to brain regions most impacted by the disease (deep cerebellar nuclei). Moreover, direct injection to the brain is a dangerous procedure and limits expression to a single brain structure whereas the FXN protein is widely expressed and known to impair cortical, cerebellar, and sensory functions.

[0012] The present invention achieves widespread transduction to both CNS and cardiac structures and cell types with a single intravenous injection, allowing for a prospect for direct benefit across all impacted modalities with a clinically tractable and safe route of administration. By allowing cells in both the CNS and cardiac tissue, by a single injection, to produce functional FXN, compositions and methods of the invention can be used to treat FXN related disorders, namely Friedrich’s ataxia.

[0013] In various embodiments, modified rAAVs are used to improve gene delivery and expression and target the CNS and cardiac tissue for gene delivery. In certain embodiments, modified rAAVs of the invention may exhibit increased specificity and transduction efficiency in the CNS and cardiac tissue, allowing for systemic delivery thereof with reduced risk of off-target effects. Such modified rAAVs may include an engineered capsid structure, acquired through iterative rounds of directed evolution and selection in non-human primates (NHPs). These engineered capsid variants may display altered tropism, with an increased specificity and Atorney Docket No.: CAPS-040 / 01WO 35847 / 188 Patent Application transduction efficiency in the CNS and cardiac tissue, and in some cases, a decreased specificity and transduction efficiency in an off-target environment. The rAAVs described herein achieve widespread transduction in the CNS (e.g., CNS cell types or tissues) and cardiac tissue in a subject upon systemic delivery (e.g., intravenous, intrathecal, intraarterial, intracranial, intraventricular, intracerebroventricular, or subcutaneous).

[0014] Aspects of the invention provide an AAV product that delivers an FXN gene supplementation or replacement strategy throughout the human CNS and cardiac tissue at levels of DNA biodistribution, RNA expression, and FXN protein levels expected to restore function in Friedrich’s ataxia patients.

[0015] Specifically, the present invention provides an adeno-associated virus (AAV) vector comprising an engineered capsid protein that includes an amino acid sequence having at least 80% shared sequence identity to ATRNGEVFIAQ (SEQ ID NO: 1). The vector further comprises (e.g. encapsidates) a nucleic acid encoding a promoter and an FXN transgene. In preferred aspects of the invention, the AAV capsid protein comprises the amino acid sequence ATRNGEVFIAQ (SEQ ID NO: 1).

[0016] Aspects of the invention provide an adeno-associated virus (AAV) vector comprising an engineered capsid protein that includes an amino acid sequence having at least 90% shared sequence identity to ATRNGEVFIAQ (SEQ ID NO: 1).

[0017] The capsid protein may be engineered from an AAV9 capsid protein (SEQ ID NO: 3). The sequence for SEQ ID NO: 3 is reproduced below: MAADGYEPDWLEDNLSEG1REWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDK GEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKK RLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPI GEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPT YNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNF KLFNIQVKEVTDNNGVKTIANNLTSTVQVFTDSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYL TLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLY YLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSW ALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVA TESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDGNFHPSPLM GGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQY TSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL Atorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent Application

[0018] For example, the capsid protein may be engineered, relative to an AAV9 capsid protein (SEQ ID NO: 3), by substitution of amino acid position 588 with the amino acid T, insertion between amino acid positions 588 and 589 of the amino acids RNGEVFI (SEQ ID NO: 2), and inclusion of parental AAV9 amino acids AQ in positions 589 and 590.

[0019] As a result, the amino acid positions 587-597 of the AAV capsid comprises the sequence ATRNGEVFIAQ (SEQ ID NO: 1).

[0020] A person of skill in the art will understand the equivalent positions of AAV vectors other than AAV9 in which the sequence ATRNGEVFIAQ (SEQ ID NO: 1) may be adapted.

[0021] Accordingly, in certain embodiments, the AAV vector may comprise an AAV9 backbone. For example, the AAV vector may comprise an AAV capsid protein comprising an amino acid sequence that is at least 98% identical to amino acid 217 to amino acid 736 of AAV9 (SEQ ID NO: 3).

[0022] Advantageously, the AAV is characterized by at least one of an increased specificity and / or increased transduction efficiency in the central nervous system (CNS) and cardiac tissue, without being bound to a mechanism of action, thereby increasing the efficacy of AAV approaches to FXN gene supplementation or replacement.

[0023] The AAV capsid protein may be characterized by at least one of an increased specificity and / or increased transduction efficiency in the brain and heart.

[0024] Further advantageously, an AAV vector targeted to the CNS and / or cardiac tissue may further comprise a nucleic acid expressing a therapeutic FXN transgene. The FXN transgene may encode a peptide having at least 95% identity to the full length (210 amino acid) FXN protein.

[0025] The nucleic acid expressing the transgene may further comprise a CAG promoter. The CAG promoter may comprise human cytomegalovirus immediate early enhancer (CMV enhancer), a chicken 0-actin promoter, and a chimeric rabbit P-globin (rBG) intron. The nucleic acid expressing the transgene may further comprise an endogenous FXN promoter, for example the miniFXNlO promoter, as described herein.

[0026] The AAV vector may further comprise at least one of the regulatory elements known to increase expression of the therapeutic gene, poly(A) signal for stability. For example, the regulatory element may be a Woodchuck hepatitis virus Post-transcriptional Regulatory Element (WPRE). The poly (A) signal is hGH poly A. Atorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent Application

[0027] Aspects of the invention also provide methods of treating a disease comprising administration of an AAV vector of the present invention. Accordingly, aspects of the invention provide a method of treatment of a disease that comprises administering a composition comprising an adeno-associated virus (AAV) vector comprising an engineered capsid protein that comprises an amino acid sequence having at least 80% shared sequence identity to ATRNGEVFIAQ (SEQ ID NO: 1), further comprising (e.g. encapsidates) a nucleic acid encoding a promoter and an FXN transgene. Methods of the invention are understood to use any of the AAV vectors described in the instant application.

[0028] Aspects of the invention provide an adeno-associated virus (AAV) vector comprising an engineered capsid protein that includes an amino acid sequence having at least 90% shared sequence identity to ATRNGEVFIAQ (SEQ ID NO: 1).

[0029] The disease treated may be a disease caused by an FXN mutation, for example Friedrich’s ataxia. In certain embodiments, the composition comprising rAAVs of the invention are administered as an injection. In certain embodiments, the composition comprising rAAVs of the invention are administered as an intravenous injection. In certain embodiments, the composition comprising rAAVs of the invention is administered as a single dose. In certain embodiments, the composition comprising rAAVs of the invention is administered as a single dose of an intravenous injection. In certain embodiments, the compositions of the invention are administered once daily.

[0030] Therapeutically effective amounts of the rAAV may be administered systemically (e.g., intracranial, intraventricular, intracerebroventricular, intravenous, intraarterial, intranasal, intrathecal, intracistemae magna administration, or subcutaneously). Advantageously, systemic administration allows for a single injection to target both CNS and cardiac tissue.

[0031] Brief Description of the Drawings

[0032] FIG. 1A-1E are graphs of WDNA biodistribution levels and RNA expression levels in non-human primate (NHP) tissues following administration of a vector of the invention.

[0033] FIG. 2A-2P are immunofluorescent images of CNS tissue in NHPs following administration of a vector of the invention.

[0034] FIG. 3 is a graph quantifying CNS tissue expression in NHPs following administration of a vector of the invention.

[0035] FIG. 4 shows chromogenic images of transgene expression in dorsal root ganglia (DRG) tissue in NHPs following administration of a vector of the invention. Atorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent Application

[0036] FIG. 5A-B shows chromogenic images of transgene expression in cardiac tissue in NHPs following administration of a vector of the invention.

[0037] FIG. 6 is a graph quantifying cardiac tissue expression in NHPs following administration of a vector of the invention.

[0038] FIG. 7 is a graph of FXN protein expression in the cardiac left ventricle and motor cortex tissue in NHPs following administration of a vector of the invention.

[0039] FIG. 8 shows images of a western blot of NHP cortex and heart tissue following administration of a vector of the invention.

[0040] FIG. 9 A and 9B are graphs of retina DNA biodistribution and RNA expression levels following direct and central IV routes of administration.

[0041] FIG. 10A-B are graphs of miniFXNlO construct performance measured by HA signal in HEK293T cells.

[0042] FIG. 11 is a graph of RNA enrichment in NHP tissues following administration of miniFXNlO and CAG promoter driven hFXN constructs of the invention.

[0043] FIG. 12 is a graph of RNA enrichment over AAV9 in NHP motor fontal cortex tissue following administration of miniFXNlO and CAG promoter driven hFXN constructs of the invention.

[0044] FIG. 13 is a graph of enrichment relative to AAV9 in NHP cardiac tissue.

[0045] FIG. 14 is a graph of DNA enrichment versus RNA enrichment in NHP cerebellar cortex tissues following administration of miniFXNlO and CAG promoter driven hFXN constructs of the invention.

[0046] FIG. 15 provides data for R5.57-CAG-hFXN driven cynomologous and human FXN expression measured by species-specific mass spectrometry.

[0047] FIG. 16A provides the data for DNA biodistribution for CAG and miniFXNlO in the CNS.

[0048] FIG. 16B provides the data for RNA expression for CAG and miniFXNlO in the CNS.

[0049] FIG. 17A provides the data for DNA biodistribution for CAG and miniFXNlO in peripheral tissues.

[0050] FIG. 17B provides the data for RNA expression for CAG and miniFXNlO in peripheral tissues. Atorney Docket No.: CAPS-040 / 01WG 35847 / 188

[0051] Patent Application

[0052] FIG. 18 provides the data for human / cyno FXN protein expression in motor cortex, cerebellar cortex, and heart by ELISA.

[0053] FIG. 19A and FIG. 19B provides data related to the expression of R5.57-CAG-hFXN- HA in the retina and optic nerve using immunocytochemistry.

[0054] FIG. 20 provides the data related to transduction in various cell types.

[0055] FIG. 21 provides the data related to ELISA experiments in HEK293 cells.

[0056] Detailed Description

[0057] The present invention provides an AAV product that delivers an FXN gene thereby implementing a supplementation or replacement strategy throughout the human CNS and heart at levels of DNA biodistribution, RNA expression, and FXN protein levels expected to restore function in Friedrich’s ataxia patients.

[0058] Friedrich’s ataxia

[0059] Friedrich's ataxia is an autosomal recessive disorder caused by a trinucleotide repeat expansion (called a GAA triplet repeat) of the frataxin (FXN) gene, on chromosome 9ql2-13, which leads to a deficiency in the mitochondrial protein frataxin (FXN) and affects 1 in 15,000 people worldwide. Frataxin is a highly conserved, 210 amino acid (~17 kDa) protein encoded in the nucleus.

[0060] The major frataxin mRNA transcript in humans (FXN-1, 1.3 kb), is composed of five exons (1 A, 2-4, and 5 A). FXN-1 mRNA encodes the full-length 210-amino acid form of protein frataxin (1-210) with a molecular weight (MW) of 23,135 Da sometimes known as isoform A or FXN-1 (Q16595-1, Uniprot). The full-length frataxin protein is rapidly translocated from the cytosol to the mitochondria where it is cleaved by mitochondrial processing peptidase. The mature biologically active form of frataxin (81-210) in mitochondria arises from atwo-step process: initial cleavage of full-length frataxin (1-210) occurs at the R-2 site between G41-L42 to give an intermediate form of frataxin (42-210), a 169-amino acid protein with a MW of 18,826 Da. The intermediate form then undergoes a second mitochondria processing peptidase-mediated cleavage atK80-S81 (a second R-2 site) to give mature frataxin (81-210), as a 130-amino acid protein with a MW of 14,268 Da. Atorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent Application

[0061] Mature frataxin is a critically important protein because it facilitates the assembly of mitochondrial iron-sulfur cluster protein complexes such as aconitase, lipoate synthase, and succinate dehydrogenases.

[0062] Although the exact role of frataxin has not been completely delineated, homozygous deletions are embryonically lethal. Evidence suggests frataxin is involved in iron metabolism, iron storage, iron-sulfur cluster formation, and protection against reactive oxygen species. Dysregulation of FXN leads to iron accumulation in the mitochondria and insufficient iron in the cytoplasm. Excess mitochondrial iron increases the incidence of iron-catalyzed reduction of hydrogen peroxide generating toxic reactive oxygen species. The increase in free radicals disrupts iron homeostasis in the mitochondria and affects the iron-sulfur cluster aconitase, a major component of cellular respiration.

[0063] Transgene delivery

[0064] 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

[0065] Transgene delivery strategies may result in gene replacements or gene supplementation, used interchangeably herein. Specifically, gene replacement refers to the 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.

[0066] 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.

[0067] 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, Atorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent Application the particular virus 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, IO11, 1012, 1013, 1014, 1015transducing units or more.

[0068] In some embodiments, the viral particle is 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. 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.

[0069] The delivery vector may be administered to the desired region(s) of the CNS by any route known in the art, including but not limited to, intrathecal, intracerebral, intra-ci sterna magna, intraventricular, intranasal, intra- aural, intra-ocular (e.g., intra-vitreous, sub-retinal, suprachoroidal, anterior chamber) and periocular (e.g., sub-Tenon's region) delivery or any combination thereof.

[0070] In some embodiments, the viral particle is administered directly to cardiac cells, e.g., intracardiac administration. Direct administration may result in high specificity of transduction of cardiac 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 cardiac cells

[0071] 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. Atorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent Application

[0072] 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). 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 intraparenchymal injection.

[0073] 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.

[0074] Advantageously, systemic administration may result in high specificity of transduction of CNS cells and cardiac 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 and cardiac cells.

[0075] 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, 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.

[0076] 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 Atorney Docket No. : CAPS-040 / 01WG 35847 / 188 Patent Application 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 and cerebrospinal fluid circulatory systems. Examples of systemic administration for the CNS and heart 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 cisterna magna). The terms “systemic administration” and “systemic delivery” are used interchangeably.

[0077] 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.

[0078] 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.

[0079] 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, or 2xl017. In some cases, the concentration of the infectious particles is 3xl07, 3xl08, 3xl09, 3xlO10, 3xlOn, 3xl012, 3xl013, 3xl014, 3xl015, 3xl016, or 3xl017. Atorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent Application

[0080] In some cases, the concentration of the infectious particles is 4xl07, 4xl08, 4xl09, 4xlO10, 4xlOu, 4xl012, 4xl013, 4xl014, 4xl015, 4xl016, or 4xl017. In some cases, the concentration of the infectious particles is 5xl07, 5xl08, 5xl09, 5xlO10, 5xlOn, 5xl012, 5xl013, 5xl014, 5xlO15, 5xl016, or 5xl017. In some cases, the concentration of the infectious particles is 6xl07, 6xl08, 6xl09, 6xlO10, 6xlOu, 6x1012, 6xl013, 6xl014, 6xl015, 6x1016, or 6xl017. In some cases, the concentration of the infectious particles is 7xl07, 7xl08, 7xl09, 7xlO10, 7xlOn, 7xl012, 7xl013, 7xl014, 7xl015, 7xl016, or 7xlO17In some cases, the concentration of the infectious particles is 8xl07, 8xl08, 8xl09, 8xlO10, 8xlOn, 8xl012, 8xl013, 8xl014, 8xlO15, 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

[0081] 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 be 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.

[0082] 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 Atorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent Application 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.

[0083] 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.

[0084] 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 are 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.

[0085] 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 Atorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent Application 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.

[0086] 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.

[0087] 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.

[0088] 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 even higher, infectious parti cles / 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.

[0089] 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. Atorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent Application

[0090] 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.

[0091] Viral Vectors

[0092] 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.

[0093] 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 encapsi dated heterologous nucleic acid, (5) convert the heterologous nucleic acid from single-stranded to double-stranded DNA as a template fortranscription 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.

[0094] 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 Atorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent Application 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).

[0095] 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.

[0096] The AAV VP3 structure contains highly conserved regions that are common to all serotypes, a core eight-stranded P-barrel motif (]3B-pi) and a small a-helix (aA). The loop regions inserted between the P-strands consist of the distinctive Hl loop between P-strands H and 1, the DE loop between P-strands D and E, and nine variable regions (VRs), which are 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.

[0097] 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) and cardiac cells. In particular, the AAV capsid proteins disclosed herein enable rAAV-mediated transduction of a heterologous nucleic acid (e.g., transgene) in the CNS and / or heart 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. Atorney Docket No.: CAPS-040 / 01WO 35847 / 188 Patent Application

[0098] The rAAV capsid proteins are engineered for optimized transduction and transgene expression in the CNS, for example the brain, and heart 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 both CNS and the heart of a subject, as compared to a reference AAV.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] The capsid protein is engineered, relative to an AAV9 capsid protein (SEQ ID NO: 3), by substituting amino acid position 588 with the amino acid T, peptide insertion between amino acid positions 588 and 589 the amino acids RNGEVFI (SEQ ID NO: 2), and parental AAV9 amino acids AQ in positions 589 and 590.

[0103] As a result, the amino acid positions 587-597 of the AAV capsid comprises the sequence ATRNGEVF1AQ (SEQ ID NO: 1).

[0104] 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 Atorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent Application 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.

[0105] 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.

[0106] 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.

[0107] 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.

[0108] 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. Atorney Docket No.: CAPS-040 / 01WP 35847 / 188 Patent Application

[0109] 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. These methods include measuring the 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.

[0110] 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.

[0111] Provided herein are methods of delivering a heterologous nucleic acid to a target in vivo environment, for example the CNS or heart. These methods comprise the composition comprising a rAAV particle with a rAAV capsid protein and 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 and cardiac cells of the subject, even when administered to the subject systemically.

[0112] Methods may comprise delivering a rAAV particle comprising an rAAV capsid protein with increased transduction and transgene expression efficiency when measured in the CNS and heart in the subject. In some embodiments, delivery is systemic. Alternatively, delivery is direct (e.g., into the affected area of the CNS or heart).

[0113] For example, exemplary AAV vectors may be described as below:

[0114] Exemplary AAV vector capsid sequence comprising ATRNGEVFIAQ (SEP ID NO: 1)

[0115] MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPG NGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGG NLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRL NFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGGAPVADNNEGADGVGSSSGNWH CDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGYSTPWGYFDFN RFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFT DSDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQML Atorney Docket No.: CAPS-040 / 01WO 35847 / 188

[0116] Patent Application

[0117] RTGNNFQFSYEFENVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTINGSGQNQQTLKFSV

[0118] AGPSNMAVQGRNYIPGPSYRQQRVSTTVTQNNNSEFAWPGASSWALNGRNSLMNPGP

[0119] AMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKVMITNEEEIKTTNPVATESYGQVA

[0120] TN HQ S ATRNGEVFIAQ AQTGW VQNQGILPGMVWQDRD VYLQGPIW AKIPHTDGNFHP S

[0121] PLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENS

[0122] KRWNPEIQYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL (SEQ ID NO: 4)

[0123] Nucleic acid sequence encoding exemplary Capsid 1

[0124] ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACAACCTTAGTGAAGGAATT

[0125] CGCGAGTGGTGGGCTTTGAAACCTGGAGCCCCTCAACCCAAGGCAAATCAACAACA

[0126] TCAAGACAACGCTAGAGGTCTTGTGCTTCCGGGTTACAAATACCTTGGACCCGGCAA

[0127] CGGACTCGACAAGGGGGAGCCGGTCAACGCAGCAGACGCGGCGGCCCTCGAGCAC

[0128] GACAAAGCCTACGACCAGCAGCTCAAGGCCGGAGACAACCCGTACCTCAAGTACAA

[0129] CCACGCCGACGCCGAGTTCCAGGAGCGGCTCAAAGAAGATACGTCTTTTGGGGGCA

[0130] ACCTCGGGCGAGCAGTCTTCCAGGCCAAAAAGAGGCTTCTTGAACCTCTTGGTCTGG

[0131] TTGAGGAAGCGGCTAAGACGGCTCCTGGAAAGAAGAGGCCTGTAGAGCAGTCTCCT

[0132] CAGGAACCGGACTCCTCCGCGGGTATTGGCAAATCGGGTGCACAGCCCGCTAAAAA

[0133] GAGACTCAATTTCGGTCAGACTGGCGACACAGAGTCAGTCCCAGACCCTCAACCAA

[0134] TCGGAGAACCTCCCGCAGCCCCCTCAGGTGTGGGATCTCTTACAATGGCTTCAGGTG

[0135] GTGGCGCACCAGTGGCAGACAATAACGAAGGTGCCGATGGAGTGGGTAGTTCCTCG

[0136] GGAAATTGGCATTGCGATTCCCAATGGCTGGGGGACAGAGTCATCACCACCAGCAC

[0137] CCGAACCTGGGCCCTGCCCACCTACAACAATCACCTCTACAAGCAAATCTCCAACAG

[0138] CACATCTGGAGGATCTTCAAATGACAACGCCTACTTCGGCTACAGCACCCCCTGGGG

[0139] GTATTTTGACTTCAACAGATTCCACTGCCACTTCTCACCACGTGACTGGCAGCGACT

[0140] CATCAACAACAACTGGGGATTCCGGCCTAAGCGACTCAACTTCAAGCTCTTTAACAT

[0141] TCAGGTCAAAGAGGTTACGGACAACAATGGAGTCAAGACCATCGCCAATAACCTTA

[0142] CCAGCACGGTCCAGGTCTTCACGGACTCAGACTATCAGCTCCCGTACGTGCTCGGGT

[0143] CGGCTCACGAGGGCTGCCTCCCGCCGTTCCCAGCGGACGTTTTCATGATTCCTCAGT

[0144] ACGGGTATCTGACGCTTAATGATGGAAGCCAGGCCGTGGGTCGTTCGTCCTTTTACT

[0145] GCCTGGAATATTTCCCGTCGCAAATGCTAAGAACGGGTAACAACTTCCAGTTCAGCT

[0146] ACGAGTTTGAGAACGTACCTTTCCATAGCAGCTACGCTCACAGCCAAAGCCTGGACC

[0147] GACTAATGAATCCACTCATCGACCAATACTTGTACTATCTCTCTAGAACTATTAACG Atorney Docket No.: CAPS-040 / 01WG 35847 / 188

[0148] Patent Application

[0149] GCAGCGGCCAAAACCAACAAACGCTAAAATTCAGTGTGGCCGGACCCAGCAACATG GCTGTCCAGGGAAGAAACTACATACCTGGACCCAGCTACCGACAACAACGTGTCTC AACCACTGTGACTCAAAACAACAACAGCGAATTTGCTTGGCCTGGAGCTTCTTCTTG GGCTCTCAATGGACGTAATAGCTTGATGAATCCTGGACCTGCTATGGCCTCTCACAA AGAAGGAGAGGACCGTTTCTTTCCTTTGTCTGGATCTTTAATTTTTGGCAAACAAGG TACTGGCAGAGACAACGTGGATGCGGACAAAGTCATGATAACCAACGAAGAAGAA ATTAAAACTACTAACCCGGTAGCAACGGAGTCCTATGGACAAGTGGCCACAAACCA CCAGAGTGCCACCAGAAACGGCGAAGTCTTCATCGCCCAAGCCCAAACCGGTTGGG TTCAAAACCAAGGAATACTTCCGGGTATGGTTTGGCAGGACAGAGATGTGTACCTGC AAGGACCCATTTGGGCCAAAATTCCTCACACGGACGGCAACTTTCACCCTTCTCCGC TGATGGGAGGGTTTGGAATGAAGCACCCGCCTCCTCAGATCCTCATCAAAAACACA CCTGTACCTGCGGATCCTCCAACGGCCTTCAACAAGGACAAGCTGAACTCTTTCATC ACCCAGTATTCTACTGGTCAAGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGA AAACAGCAAGCGCTGGAACCCGGAGATCCAGTACACTTCCAACTATTACAAGTCTA ATAATGTTGAATTTGCTGTTAATACTGAAGGTGTATATAGTGAACCCCGCCCCATTG GCACCAGATACCTGACTCGTAATCTG (SEQ ID NO: 5)

[0150] Promoter / Enhancers

[0151] 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.

[0152] 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 / enhancer element may be constitutive or inducible.

[0153] Promoters are DNA regions that initiate gene transcription by controlling the binding of RN A 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 Atorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent Application 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, a human synapsin I promoter, and an endogenous FXN promoter, for example the miniFXNlO promoter as described herein. 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. 2011 Sep; 22(9): 1143-1153; Glover et al., Adenoviral-mediated, High-Level, Cell-Specific Transgene 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; Li et al. (2020) “Defining Transcription Regulatory Elements in the Human Frataxin Gene: Implications for Gene Therapy” Hum Gene Ther. 31(15-16 :839-85,1 the content of each of which is incorporated herein by reference.

[0154] 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 tissuespecific 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 Atorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent Application elements for oligodendrocytes such as, but not limited to, the myelin basic protein (MBP) promoter.

[0155] 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 are considered when selecting a PRE.

[0156] Examples

[0157] Construct 1

[0158] Construct 1 was engineered and developed as a gene replacement therapy product to be administered as a single intravenous (IV) injection to FA patients. Construct 1 consists of a novel capsid designed to deliver the functional FXN gene to neurons in the CNS, cardiomyocytes, and sensory neurons, thereby providing a permanent source of FXN protein allowing for restoration of cellular respiration and prevention of cell death. Gene therapy specifically offers the potential to stably replace wild type FXN protein with a single administration, enabling long-term disease modification and substantially slowing disease progression with limited treatment burden.

[0159] Construct 1 consists of a recombinant adeno-associated virus (rAAV) of the invention engineered to enrich delivery of cargo containing the FXN gene. The rAAVs of the invention disclosure were designed as a modified adeno-associated virus serotype 9 capsid containing an amino acid substitution of T in AA588 and 7-mer insertion (RNGEVFI, SEQ ID NO: 2) between AA588 and AA589 (together, ATRNGEVFIAQ, SEQ ID NO: 1, are the resulting residues at positions 587-597) to the rAAV capsid protein and has been engineered to deliver the human frataxin protein to the CNS and heart following a single intravenous injection.

[0160] The cargo / payload of the vector (CAG-FXN-WPRE-bGH) includes a CAG promoter with 3 regulatory elements: human Cytomegalovirus immediate early enhancer (CMV enhancer), a chicken beta-actin promoter, and a chimeric rabbit 0-globin (rBG) intron, altogether named CAG. The payload further includes the wild-type human FXN transgene (NCBI reference sequence Atorney Docket No.: CAPS-040 / 01WO 35847 / 188 Patent Application

[0161] NP_000135.2), a Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and a bovine growth hormone polyadenylation signal (bGH-polyA). An iteration of the cargo composition includes an HA epitope tag at the C-terminus of the FXN protein (HA sequence: YPYDVPDYA).

[0162] Study 1: Single dose administration and quantification

[0163] Friedreich’s Ataxia (FA) is a rare, hereditary, form of ataxia with no approved disease modifying treatment. FA is caused by an intronic triplicate repeat expansion that diminishes the expression of the frataxin (FXN) protein causing progressive deficits in cellular respiration and cell death, with pronounced impact to functions controlled by non-regenerative cell types (neurons, cardiomyocytes).

[0164] A study was conducted to assess performance of Construct 1 after systemic administration in 3 male cynomologus macaques (Macaco, fascicularis) aged 33-34 months of age. Animals were administered HA-tagged Construct 1 at a dose of 2.5E13 vg / kg as a single intravenous (IV) infusion with necropsy following a 4-week in-life duration.

[0165] Downstream analyses included DNA and RNA biodistribution of the CNS and peripheral organs, quantification of transduction in key organs and cell types using histological immunostaining of the HA tag, and quantification of bulk FXN protein levels in the brain and heart by ELISA and Western Blot.

[0166] CNS and neuronal tissue expression

[0167] FIG. 1A-1E are graphs of FAVDNA biodistribution levels and RNA expression levels in non-human primate (NHP) tissues following administration of Construct 1. Tissues included the motor cortex, cerebellum, cervical spinal cord (SC), lumbar SC, thoracic SC, left ventricle, dorsal root ganglia (DRG), and liver. At the DNA level, Construct 1 was ~6x de-targeted from the liver and about ~4x de-targeted to DRGs compared to AAV9 administered at the same dose.

[0168] Overall, Construct 1 achieves therapeutically meaningful CNS / PNS RNA expression levels in key FA tissues while de-targeting liver.

[0169] FIG. 2A-2P are immunofluorescent images of CNS tissue following administration of HA- tagged Construct 1.

[0170] FIG. 3 is a graph quantifying the percentage of neurons expressing the HA tag. Transduction rates in CNS target regions and cell types, including motor neurons (motor cortex), Atorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent Application

[0171] Purkinje cells, dentate nucleus, and motor neurons (spinal cord), achieve greater than 80% expression following administration of Construct 1.

[0172] FIG. 4 shows chromogenic images of transgene expression in DRG tissue following administration of HA-tagged Construct 1. DRGs are one of the major sites of pathology in Friedrich’s ataxia. However, in most patients DRG cells never develop or are degenerated at the time of diagnosis. Construct 1 transduced DRGs and may provide therapeutic benefit as younger patients are identified.

[0173] FIG. 15 provides data related to the species-specified targeted mass spectrometry for human FXN expression in both the motor cortex and heart of NHPs. Species-specific mass spectrometry assay differentiates endogenous (cynomolgus) FXN protein from the human ortholog encoded by the viral vector provided herein. Each line in FIG. 18 connects the endogenous FXN protein (cFXN) level measured in an NHP tissue sample to the sum of the endogenous and human FXN (cFXN + hFXN) protein level measured in the same tissue sample.

[0174] As the data provided in FIG. 15 provides that following administration of R5.57-CAG- hFXN-WPRE-HA at 2.5E13 vg / kg, hFXN protein expression levels were on average 9x higher than endogenous levels in the motor cortex and 1.35x higher than endogenous levels in the left ventricle.

[0175] Cardiac tissue expression

[0176] FIG. 5A-B shows chromogenic images of transgene expression in cardiac tissue following administration of HA-tagged Construct 1.

[0177] The construct delivers therapeutically meaningful cardiac transduction.

[0178] FIG. 6 is a graph quantifying the overall area in the left ventricle expressing the HA tag. Transduction rates in cardiac target regions reached 27% coverage in the cardiac left ventricle.

[0179] Cardiac and CNS expression

[0180] FIG. 7 is a graph of FXN protein expression in the cardiac left ventricle and motor cortex following administration of a vector of the invention as measured by ELISA. FXN protein expression was 1.7x higher than endogenous levels in the cardiac left ventricle and 8.2x higher than endogenous levels in the motor cortex. At 2.5E13 vg / kg, this exceeds the putative efficacy threshold of 30% over endogenous levels in the CNS and cardiac tissue while also below the putative safety cutoff of 9x higher than endogenous levels in the cardiac left ventricle. Atorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent Application

[0181] As described above, full-length FXN contains an N-terminal mitochondrial targeting sequence, which is cleaved in the mitochondria by mitochondrial processing peptidase to generate mature FXN (81-210).

[0182] FIG. 8 shows images of a western blot of NHP cortex and heart tissue following administration of Construct 1. Detection of HA mature FXN suggests AAV-delivered FXN is properly localizing to the mitochondria.

[0183] Retinal expression

[0184] Greater than 30% of FA patients experience blindness over the course of disease due to degeneration of retinal ganglion cells.

[0185] FIG. 9A and 9B are graphs of retina DNA biodistribution and RNA expression levels following direct (SCS-suprachoroidal space and IVT-intravitreal) and central (IV-intravenous) routes of administration.

[0186] Exposure to the retina by Construct 1 from central IV administration resulted in RNA expression levels similar with both direct SCS and IVT administration as previously measured in an NHP pool of 20 AAV2 and AAV9 variants.

[0187] Construct 1 demonstrated target delivery of the human Frataxin transgene to the CNS, heart, and sensory neurons with widespread detection of vector DNA and RNA in the brain, spinal cord, DRGs, retina and left ventricle. Simultaneously, there was reduced detection of vector DNA and RNA in the liver. Exposure to the retina could represent significant improvement in quality of life and differentiation from the field.

[0188] Transduction levels were >80% in all CNS regions and cell types of interest. FXN protein expression exceeded 30% target expected to provide prospect for direct benefit based on human genetics, with a 70% increase in the heart and 800% increase in the motor cortex. Detection of all 3 FXN protein isoforms by Western Blot confirms appropriate subcellular localization of Construct 1 delivered FXN protein.

[0189] The dose tested was well tolerated at 4-weeks and considered a no observed adverse effect level per consultants and histopathologist.

[0190] Altogether, results showed widespread expression in the brain and heart at both nucleic acid and protein levels expected to provide a prospect for direct benefit in Friedreich’s ataxia patients.

[0191] Study 2: Dose-dependent vector administration and quantification Atorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent Application

[0192] A study was conducted to assess Construct 1 -driven dose-dependent increase in FXN protein after systemic injection with the surrogate capsid CAP-BIO in wild-type mice. Mice were administered either lEl lvg / kg or 1E12 vg / kg of CAP-B10:CAG-FXN-HA-WPRE-bGEI-HA via a single retroorbital IV injection at 2-months of age with necropsy following a 4 week in-life duration. A group of untreated animals were euthanized at 3 months of age as controls. Downstream analysis included quantification of bulk viral-induced FXN and endogenous mFXN expression by ELISA.

[0193] A dose-dependent increase in FXN expression was observed in the heart, cerebellum, and cerebrum achieving levels that are expected to provide a prospect for direct benefit in FXN deficient models and patients.

[0194] Discussion

[0195] Leveraging the high throughput directed evolution engineering platform, the present invention has identified a systemically administered (IV) capsid that achieves NHP brain-wide biodistribution transducing large percentages of neurons in key FA-related brain areas in addition to delivering therapeutically significant levels of cardiac transduction.

[0196] In Study 1, dosing a small pool of capsids simultaneously (N=3 NHP), the Construct 1 drives RNA expression levels ~100x higher than AAV9 in CNS, while maintaining similar RNA expression in cardiac tissue, and ~10x de-targeting in the liver. When administered IV as a single variant at a low to moderate dose (N=3 NHP), Construct 1 delivering FXN transduced more than 80% of cerebellar Purkinje cells, dentate nucleus neurons, motor neurons in the cortex and spinal cord, and nearly 30% of cardiac left ventricle tissue area, on average. Bulk protein levels in treated NHPs were 1.7x higher than endogenous levels in the left ventricle, and 8.2x higher than endogenous levels in the motor cortex by ELISA. Moreover, significant RNA expression levels were detected in the retina (~1E6 copies / ug RNA) by qPCR suggesting a potential benefit for sensory vision loss experienced by FA patients. Significant de-targeting of the liver and other nontarget tissues contributed to the favorable safety profile characterized by no adverse immunogenicity, clinical pathology, and histopathology findings. Together, these data demonstrate that a drug product driven by Construct 1 produces therapeutically meaningful FXN expression in CNS, cardiac, and sensory regions impacted by disease and has the potential to become a best-in-class targeted therapy for the treatment of FA. Atorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent Application

[0197] The rAAVs of the invention deliver the FXN gene supplementation or replacement strategy throughout the human CNS and heart at levels of DNA biodistribution, RNA expression, and FXN protein levels expected to restore function in Friedrich’s ataxia patients, demonstrated to provide an increase in the efficacy shown by previous AAV approaches to FXN gene supplementation or replacement based on the preclinical NHP studies.

[0198] Given the absence of targeted, disease modifying therapies and only modest efficacy of current available therapies, Construct 1 has the potential to be a practical and feasible therapy that addresses significant unmet needs in FA patients.

[0199] Study 3: miniFXNl 0-hFXN enrichment

[0200] Endogenous FXN expression cassettes were designed and screen in vitro and in vivo to identify a single construct for further development in DC-enabling studies.

[0201] Expressions cassettes were designed based on Li et al. (2020) “Defining Transcription Regulatory Elements in the Human Frataxin Gene: Implications for Gene Therapy” Hum Gene Ther. 31(15-16 :839-851, incorporated by reference in its entirety herewith.

[0202] 12 expression cassettes were screened in vitro to identify 4 cassettes that were included in an NHP pool. An in vivo pool was used to identify top constructs to advance for further development. 3 cynomolgus macaques were injected with a pool containing 26 constructs at a dose of 1.25E13 vg.kg for a 32 day in-life study.

[0203] FIG. 10A-B are graphs of miniFXNlO construct performance measured by HA signal in HEK293T cells.

[0204] The endogenous promoter miniFNXIO was chosen based on results of the in vivo pool. The miniFXNlO promoter is a functional shortened endogenous promoter that comprises a combination of elements from constructs described in Li et al. 2020. The miniFXNlO promoter comprises a 246 bp 5’ UTR from construct 10 of Li et al. 2020 combined with a 297bp partial intron from construct 8 in Li et al.

[0205] CAG-hFXN and mini -FXN10-hFXN were paired with three different capsids (Capsid 1, Capsid 2, Capsid 3) and the constructs were administered to NHPs.

[0206] FIG. 11 is a graph of RNA enrichment in NHP tissues following administration of miniFXNlO and CAG promoter driven hFXN constructs of the invention.

[0207] Capsid 1 -miniFXNl 0-hFXN constructs achieved reduced RNA expression relative to Capsid 1 -CAG-hFXN constructs. Constructs with the miniFXNlO promoter elements achieved Atorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent Application reduced RNA expression in FA target regions and liver, showing 14%-26% of the CAG construct’ s RNA enrichment in FA target regions and 40-60% in the liver.

[0208] FIG. 12 is a graph of RNA enrichment over AAV9 in NHP motor fontal cortex tissue following administration of miniFXNlO and CAG promoter driven hFXN constructs of the invention.

[0209] FIG. 13 is a graph of enrichment relative to AAV9 in NHP cardiac tissue.

[0210] Cardiac RNA enrichment is in line with levels achieved by other AAV serotypes currently undergoing clinical testing for treatment of FA-related cardiomyopathy.

[0211] Enrichment ratios are shown in the table below:

[0212] The pool data suggests that a functional shortened endogenous promoter (miniFXNlO) regulates FXN expression levels. miniFXNlO-hFXN enrichment maintained the same level with CAG-hFXN enrichment increased with improved capsid performance suggesting that miniFXNlO provides regulation.

[0213] Capsid 2-miniFXNl 0-hFXN RNA enrichment was identical to Capsid 1-miniFXNlO- hFXN enrichment, while Capsid 2-CAG-hFXN RNA enrichment was 1.4x of Capsid 1-CAG- hFXN RNA enrichment. Capsid 3-miniFXNl 0-hFXN RNA enrichment was 0.93x of Capsid 2- miniFXNl 0-hFXN enrichment, while Capsid 3-CAG-hFXN RNA enrichment was 1.24x Capsid 2-CAG-hFXN RNA enrichment.

[0214] FIG. 14 is a graph of DNA enrichment versus RNA enrichment in NHP cerebellar cortex tissues following administration of miniFXNlO and CAG promoter driven hFXN constructs of the invention. Only data from seronegative animals is shown. Each dot represents one barcode of one animal.

[0215] RNA enrichment did not change as a function of DNA enrichment for the miniFXNlO promoter constructs. CAG promoter constructs increased RNA enrichment with increased DNA enrichment.

[0216] Discussion Atorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent Application miniFXNlO demonstrated stable RNA enrichment across a range of capsids with differing performance, while CAG-hFNX RNA enrichment increased with increasing capsid performance across the same set of capsids.

[0217] Constructs of the invention results in near complete coverage of key sites of CNS pathology. Cardiac transduction profdes exceed efficacy thresholds and exhibited a well-tolerated safety profile in NHPs over 4 weeks in-life.

[0218] Study 4: Dose range finding study

[0219] The dose range finding study dosed two constructs (provided below) with a different promoter at two different doses.

[0220] The summary of the data from the dose ranging provided below:

[0221] R5.57-CAG-hFXN-HA-WPRE

[0222] The data demonstrated robust DNA and RNA biodistribution (measured by ddPCR) across CNS and cardiac tissues. In addition, the dose-dependent increase in bulk FXN protein levels were measured by ELISA. Specifically, the studies found approximately 1.8-2x increase in cardiac tissue and approximately 2.4-5.7x increase in CNS tissues.

[0223] In addition, the data also demonstrated moderate expression in retinal ganglion cells and optic nerve by IHC. In addition, there was robust expression in upper and lower motor neurons, Purkinje cells, neurons in the dentate nucleus, and cardiac tissue.

[0224] R5.57-miniFXN10-hFXN-HA-WPRE:

[0225] The data demonstrated robust DNA biodistribution (measured by ddPCR) with modest RNA expression. This suggests that there is self-regulation. This is further bolstered by the data demonstrating no increase in bulk FXN protein levels by ELISA. Furthermore, this construct results in minimal expression in retinal ganglion cells and optic nerve, demonstrated by IHC. In addition, the data for this construct results in detectable expression in upper motor neurons and Purkinje cells by IHC. Atorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent Application

[0226] FIG. 16A provides the data related to DNA biodistribution across various CNS tissues for the two constructs and two difference concentration. The data demonstrates widespread DNA biodistribution across groups. FIG. 16B provides the data related to RNA expression across the various CNS tissues with the two constructs tested at two different concentrations. The lower expression with miniFXNlO-FXN-HA-WPRE construct suggests a possibility of self regulation of the expression.

[0227] FIG. 17A provides the data related to the DNA biodistribution in heart left ventricle, heart right ventricle, quadriceps, and sciatic nerve, for the two constructs and the two different concentrations. FIG. 17B provides the data related to RNA expression in heart left ventricle, heart right ventricle, quadriceps, and sciatic nerve, for the two constructs and the two different concentrations. As evident from the data provided in FIG. 17A and FIG. 17B, the RNA expression is weaker in miniFXNlO-FXN-HA-WPRE construct.

[0228] FIG. 18 provides the data related to dose-dependent change in the human FXN protein expression measured by ELISA. FIG. 20 provides the imaging data demonstrating the wide-spread transduction in critical cell types for both the CAG dosing groups. As evident from the data in FIG. 18, there is a dose-dependent increase in human FXN protein expression with the CAG construct, but the dose-dependent increase in expression is not observed with miniFXNlO promoter. The dose-dependent increase in CAG groups achieve significant increase in FXN protein expression in CNS (motor cortex and cerebellar cortex) and heart. In the miniFXNlO groups, the data demonstrates stable levels of FXN protein, which is consistent with regulation.

[0229] The data provided in FIG. 19A and FIG. 19B demonstrate the expression of the CAG promoter in retinal ganglion cells (RGCs), retinal nerve fiber layer (RNFL), and optic nerve. The patients suffering from FA experience gradual vision loss, which is attributed to decreased FXN expression in the retinal ganglion cells. HA staining (purple - near the top of FIG. 19A and on the top and left borders of FIG. 19B) is observed in retinal cell types, including RGCs, RNFL, and optic nerve in both CAG and miniFXN construct dose groups. However, the expression in retinal cells is much more pronounced in CAG group.

[0230] Even mild-moderate expression levels have the potential to slow progression / prevent vision loss in patients with FA. In addition, the expression of FXN was also noted in other cell types includingciliary body.

[0231] Discussion Atorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent Application

[0232] The constructs of the invention, including the CAG and miniFXNlO constructs, demonstrate the broad DNA biodistribution and RNA expression at both the doses evaluated for the constructs. The constructs have some differing properties, including the potential for selfregulation, which is further evaluated by in vitro experiments.

[0233] Constructs of the invention results in near complete coverage of key sites of CNS pathology at the tested doses. Cardiac transduction profdes exceed efficacy thresholds and exhibited a well -tolerated safety profile in NHPs over 20 weeks in-life. In addition, the constructs demonstrated expression in the ocular cells and tissues. Thus, the constructs of the invention have a potential to reduce progression of vision loss and / or treat vision loss in FA patients.

[0234] Study 5: In vitro experiments for testing self -regulation:

[0235] Experiments were conducted in HEK293T cells to evaluate the potential for selfregulation with the miniFXNlO construct. In some experimental conditions, endogenous levels of FXN were knocked down (KD) using an siRNA directed against a region in the 3’UTR of the endogenous FXN sequence. FXN protein expression measured by ELISA is reported across 3 doses of either CAG-FXN or miniFXNlO-FXN in FIG. 21. The data provided in FIG. 21 demonstrates potential for self-regulation of the miniFXNlO cargo. miniFXNlO promoter efficiently drives FXN expression and limits overexpression through self-regulation as demonstrated by -100% endogenous protein levels achieved at high doses in FXN knockdown cells and across all doses in WT HEK293T cells. This self-regulation is a beneficial property of the miniFXNlO construct since it would reduce the risks and sideeffects associated with the overexpression of FXN. This expands the therapeutic window of the constructs of the current invention.

[0236] Incorporation by Reference

[0237] 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.

[0238] Equivalents

[0239] 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 Attorney Docket No.: CAPS-040 / 01WG 35847 / 188

[0240] Patent Application 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

1. Atorney Docket No.: CAPS-040 / 01WO 35847 / 188Patent ApplicationClaims1. An adeno-associated virus (AAV) vector comprising: an engineered capsid protein comprising the amino acid sequence having at least 80% shared sequence identity to ATRNGEVF1AQ (SEQ ID NO: 1); and a nucleic acid encoding: a promoter; and an FXN transgene.

2. The AAV of claim 1, wherein the AAV capsid protein comprises the amino acid sequence ATRNGEVFIAQ (SEQ ID NO: 1).

3. The AAV of claim 1, wherein the capsid protein is engineered from an AAV9 capsid protein (SEQ ID NO: 3).

4. The AAV of claim 3, wherein the capsid protein is engineered, relative to an AAV9 capsid protein (SEQ ID NO: 3), by substitution of amino acid position 588 with the amino acid T, peptide insertion between amino acid positions 588 and 589 of the amino acids RNGEVFI (SEQ ID NO: 2), wherein parental AAV9 amino acids AQ are present at positions 589 and 590.

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

6. The AAV 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 and heart.

7. The AAV of claim 1, wherein the transgene is a wild-type human FXN transgene.

8. The AAV of claim 7, wherein the FXN transgene encodes a peptide having at least 95% identity to the full FXN protein (1-210).Atorney Docket No.: CAPS-040 / 01WO 35847 / 188Patent Application9. The AAV of claim 1, wherein the promoter is a CAG promoter.

10. The AAV of claim 9, wherein the CAG promoter comprises human cytomegalovirus immediate early enhancer (CMV enhancer), a chicken P -act in promoter, and a chimeric rabbit P- globin (rBG) intron.

11. The AAV of claim 10, wherein the AAV further comprises at least one regulatory element and / or a poly (A) signal.

12. The AAV of claim 11, wherein the regulatory element is a Woodchuck hepatitis virus Post- transcriptional Regulatory Element (WPRE).

13. The AAV of claim 11, wherein the poly(A) signal is hGH polyA.

14. A method of treatment of a disease, the method comprising administering a composition comprising adeno-associated virus (AAV) vector comprising: an engineered capsid protein comprising an amino acid sequence having at least 80% shared sequence identity to ATRNGEVFIAQ (SEQ ID NO: 1); a nucleic acid encoding: a promoter; and an FXN transgene.

15. The method of claim 14, wherein the AAV capsid protein comprises the amino acid sequence ATRNGEVFIAQ (SEQ ID NO: 1).

16. The method of claim 14, wherein the capsid protein is engineered from an AAV9 capsid protein (SEQ ID NO: 3).

17. The method of claim 16, wherein the capsid protein is engineered, relative to an AAV9 capsid protein (SEQ ID NO: 3), by substitution of amino acid position 588 with the amino acidAtorney Docket No.: CAPS-040 / 01WG 35847 / 188 Patent ApplicationT, peptide insertion between amino acid positions 588 and 589 of the amino acids RNGEVFI (SEQ ID NO: 2), wherein parental AAV9 amino acids AQ are present at positions 589 and 590.

18. The method of claim 17, wherein the AAV is characterized by at least one of an increased specificity and / or increased transduction efficiency in the central nervous system (CNS) and / or cardiac tissue.

19. The method of claim 18, wherein the AAV capsid protein is characterized by at least one of an increased specificity and / or increased transduction efficiency in the brain and heart.

20. The method of claim 14, wherein the transgene is a wild-type human FXN transgene.21 . The method of claim 20, wherein the NW transgene encodes a peptide having at least 95% identity to the full length FXN protein.

22. The method of claim 14, wherein the promoter is a CAG promoter.

23. The method of claim 22, wherein the CAG promoter comprises human cytomegalovirus immediate early enhancer (CMV enhancer), a chicken 0-actin promoter, and a chimeric rabbit 0- globin (rBG) intron.

24. The method of claim 14, wherein the AAV further comprises at least one regulatory element and / or a poly (A) signal.

25. The method of claim 24, wherein the regulatory element is a Woodchuck hepatitis virus Post-transcriptional Regulatory Element (WPRE).

26. The method of claim 24, wherein the poly(A) signal is hGH polyA.

27. The method of claim 14, wherein the disease is a disease caused by an W mutation.Atorney Docket No.: CAPS-040 / 01WG 35847 / 188Patent Application28. The method of claim 27, wherein the disease is Friedrich’s ataxia.

29. The method of claim 14, wherein the composition is administered as an intravenous injection.

30. The method of claim 29, wherein the composition is administered as a single intravenous injection.

31. The AAV of claim 1, wherein the promoter is a miniFXNlO promoter.

32. The method of claim 14, wherein the promoter is a miniFXNlO promoter.

Images