Gene therapy for ocular manifestations of CLN2 disease

Abstract

Described are compositions and methods for delivery of therapeutic products (e.g., therapeutic proteins (e.g., antibodies), therapeutic RNAs (e.g., shRNA, siRNA, and miRNA), and therapeutic aptamers) to the retina / vitreous humor of the eye of a human subject to treat ocular conditions, including, for example, recombinant viral vectors such as recombinant adeno-associated viral (rAAV) vectors.

Description

[Technical Field]

[0001] Priority This application claims the benefit of priority to U.S. Serial No. 63 / 088,911, filed October 7, 2020, and U.S. Serial No. 63 / 146,134, filed February 5, 2021, each of which is incorporated by reference in its entirety.

[0002] Reference to electronically submitted sequence listing This application incorporates by reference the Sequence Listing submitted herewith as a text file entitled "12656-144-228_Sequence_Listing.txt", created on September 30, 2021, and 42,746 bytes in size. [Background technology]

[0003] 1. Background technology Neuronal ceroid lipofuscinosis (NCL) is a group of rare, inherited neurodegenerative disorders. They are considered the most common of neurogenetic storage diseases and are associated with the accumulation of autofluorescent lipopigments resembling ceroid and lipofuscin in patients. NCL is associated with variable but progressive symptoms, including abnormally increased muscle tone or spasms, blindness or visual impairment, dementia, lack of muscle coordination, intellectual disability, movement disorders, seizures, and an unsteady gait. The disease occurs in approximately 1 in 12,500 people. There are three major types of NCL: adult (Kufs or Parry disease), juvenile, and late childhood (Jansky-Bielschowsky disease). Neuronal ceroid lipofuscinosis (NCL) was originally defined by its age of onset and clinical symptoms (as described herein). However, it has since been reclassified based on newer molecular findings, which provide evidence of a much larger overlap of distinct genetic variants than previously suggested by the clinical phenotype.

[0004] At least 20 genes have been identified in association with NCL. Patients with NCL with CLN2 mutations are deficient in a pepstatin-insensitive lysosomal peptidase called tripeptidyl peptidase 1 (TPP1). TPP1 removes tripeptides from the N-terminus of polypeptides. Mutations have been reported in all 13 exons of the CLN2 gene. Some mutations result in a more protracted course. Onset is usually in late childhood, although later onset has been described. More than 58 mutations have been described in CLN2.

[0005] CLN2 disease, a form of Batten disease, is a rare lysosomal storage disorder (LSD) with an estimated incidence of 0.07–0.51 per 100,000 live births (Augestad et al., 2006; Claussen et al., 1992; Mole et al., 2013; National Batten Disease Registry; Poupetova et al., 2010; Santorelli et al., 2013; Teixeira et al., 2003). CLN2 disease is a fatal autosomal recessive neurodegenerative LSD caused by mutations in the CLN2 gene, which is located on chromosome 11q15 and encodes the soluble lysosomal enzyme tripeptidyl-peptidase-1 (TPP1). Mutations in the CLN2 gene and subsequent deficiency of TPP1 enzyme activity result in lysosomal accumulation of storage materials and neurodegeneration in the brain and retina (Liu et al., 1998; Wlodawer et al., 2003). CLN2 disease is characterized by early onset between the ages of 2 and 4 years and typically includes early features including recurrent seizures (epilepsy) and difficulty with coordination (ataxia). The disease also results in the loss of previously acquired skills (developmental regression). Epilepsy is often refractory to medication, and a general decline in psychomotor function is rapid and uniform between the third and fifth birthdays before early death by mid-childhood (Schulz et al., 2013) (Nickel M et al., 2016; Worgall et al., 2007).

[0006] Retinal degeneration associated with classic neuronal ceroid lipofuscinosis type 2 (CLN2) disease manifests as bilateral, progressive, and symmetrical loss, beginning in the central macula in a bull's-eye pattern and extending to the peripheral retina in end-stage disease (Orlin et al., 2013 PLoS One. 2013 Aug 28;8(8):e73128). Retinal degeneration is characterized by a rapid loss of central retinal thickness (CRT) on optical coherence tomography (OCT) over a critical 2-year period (Kovacs et al., 2020; Ophthalmol Retina. 2020;4(7):728-36). However, a less common attenuated phenotype with delayed visual loss can manifest as late as adolescence (Kohlschutter et al., 2019).

[0007] Enzyme replacement therapy (ERT) using recombinant TPP1 (Brineura® cerliponase alfa, BioMarin Pharmaceuticals) was recently approved in the United States (US) and European Union (EU) for the treatment of CLN2 disease and is administered as a biweekly infusion into the lateral ventricle via a permanently implanted device. While the FDA specified that the clinical benefit of Brineura® is limited to stabilizing motor function, the European Medicines Agency (EMA) determined that it also positively impacts language skills (Brineura®, FDA New Drug Approval Review Summary, Brineura® European Published Pharmaceuticals Review Report [EPAR], Schulz et al., 2016). Brineura® requires specialized expertise to implant a port directly into the brain and must be administered in a clinical setting during a 4-hour infusion every 2 weeks by trained professionals familiar with intracerebroventricular (ICV) administration. The need for repeated infusions is due, in part, to the short CSF and lysosomal half-lives of Brineura®, estimated at 7 hours and 11.5 days, respectively (Brineura®, EPAR). Thus, there remains a significant unmet need for new therapies that can provide durable and long-term TPP1 enzyme activity in the central nervous system (CNS) of patients with CLN2 disease without the high patient burden and morbidity associated with repeated administration of ERT. Therefore, there is a need for compositions useful for delivering and expressing TPP1 in subjects requiring treatment for CLN2 disease. A single administration of recombinant adeno-associated virus (rAAV) expressing canine TPP1 (rAAV2.caTPP1) has been shown to result in high expression of TPP1, primarily in epithelial cells, secreting the enzyme into the cerebrospinal fluid and providing clinical benefit. See Katz et al, Sci Transl Med. 2015 Nov 11;7(313):313ra180, and KATZ, et al, Gene therapy 2017 Feb 24(4):215-223, which are incorporated herein by reference.However, AAV2 does not penetrate the brain parenchyma and does not target neurons, limiting the expected benefits compared to those achievable with novel neurotropic AAVs.

[0008] In late infantile neuronal ceroid lipofuscinosis (CLN2) disease, mutations in the CLN2 gene lead to a lack of TPP1 enzyme activity, resulting in severe neurodegeneration within the central nervous system and retina. A vast number of eye diseases and disorders associated with ocular pathology can be attributed to genetic alterations or protein dysregulation (Stone et al., 2017, Ophthalmology 124(9):1314-1331). Recent advances in genomics and proteomics have significantly impacted our understanding of the disease mechanisms and / or genetic basis underlying such eye diseases or conditions. Gene therapy has been employed in the treatment of certain eye diseases (see, e.g., International Patent Application No. PCT / US2017 / 027650 (Publication No. WO2017 / 181021A1)).

[0009] There are currently no approved therapies to treat the ocular symptoms of CLN2 disease, and vision loss represents a significant unmet need. With no available treatment options, all children with CLN2 disease will experience macular degeneration, which is associated with vision loss and progression to blindness (Kovacs et al., 2020; Williams et al., 2017; Nickel et al., 2018). Prior to and since the initiation of ERT for the treatment of CLN2 disease, leading research institutions have collected natural history data to understand the progression of retinal degeneration leading to vision loss in the CLN2 population. It has been found that 1) ERT does not alter the trajectory of CLN2 disease-associated retinal degeneration, 2) the onset of retinal degeneration is bilateral, affecting both eyes simultaneously and to the same extent, and 3) retinal degeneration progresses symmetrically between eyes over time. Summary of the Invention

[0010] 2. Summary of the invention In one aspect, provided herein is a method of treating an ocular symptom associated with CLN2 Batten disease in a subject in need thereof, the method comprising administering to the eye of the subject a recombinant adeno-associated virus (rAAV) comprising a capsid and a vector genome, wherein the rAAV is AAV9, and the vector genome comprises (a) an AAV 5' inverted terminal repeat (ITR), (b) a promoter, (c) a CLN2 coding sequence encoding a human tripeptidyl peptidase 1 (TPP1) protein, and (d) an AAV 3' ITR. In some embodiments, the AAV 5' ITR and / or the AAV 3' ITR are derived from AAV2. In some embodiments, the coding sequence of (c) is a codon-optimized human CLN2 that is at least 70% identical to the native human coding sequence of SEQ ID NO:2. In some embodiments, the coding sequence of (c) is SEQ ID NO:3.

[0011] In some embodiments, the promoter is a chicken beta actin promoter, hi some embodiments, the promoter is a hybrid promoter comprising a CBA promoter sequence and a cytomegalovirus enhancer element.

[0012] In some embodiments, the vector genome further comprises a poly A. In some embodiments, the poly A is synthetic or derived from bovine growth hormone (bGH), human growth hormone (hGH), SV40, rabbit β-globin (RGB), or modified RGB (mRGB).

[0013] In some embodiments, the vector genome further comprises an intron, hi some embodiments, the intron is derived from CBA, human beta globin, IVS2, SV40, bGH, alpha-globin, beta-globin, collagen, ovalbumin, or p53.

[0014] In some embodiments, the vector genome further comprises an enhancer, hi some embodiments, the enhancer is a CMV enhancer, an RSV enhancer, an APB enhancer, an ABPS enhancer, an alpha mic / bik enhancer, a TTR enhancer, en34, or ApoE.

[0015] In some embodiments, the vector genome is about 3 kilobases to about 5.5 kilobases in size, hi some embodiments, the vector genome is about 4 kilobases in size.

[0016] In some embodiments, 2×10 10 In some embodiments, 6 x 10 genome copies / eye of rAAV are administered. 10 Administer rAAV genome copies / eye.

[0017] In some embodiments, the subject has a change from baseline in central retinal thickness (CRT) as measured by SD-OCT. In some embodiments, the subject has a change from baseline in pupillary light reflex as measured by pupillometry. In some embodiments, the subject has a change from baseline in macular thickness / volume as measured by SD-OCT. In some embodiments, the subject has a change from baseline in outer nuclear layer (ONL) thickness as measured by SD-OCT. In some embodiments, the subject has a change from baseline in total retinal thickness as measured by SD-OCT. In some embodiments, the subject has a change from baseline in inner nuclear layer as measured by SD-OCT. In some embodiments, the subject has a change from baseline in photoreceptor (PR) + retinal pigment epithelium (RPE) as measured by SD-OCT. In some embodiments, the subject has a change from baseline in outer segment + RPE (OS+RPE) as measured by SD-OCT. In some embodiments, the subject has a change from baseline in ellipsoid zone (EZ) as measured by SD-OCT.

[0018] In some embodiments, the subject has a delayed onset of retinal deterioration compared to comparable clinical progression with standard of care, hi some embodiments, the subject has a delayed onset of vision loss compared to comparable clinical progression with standard of care.

[0019] In some embodiments, the method results in detectable TPP1 expression levels in the vitreous humor and / or aqueous humor of the subject's eye within 3 months of administering the rAAV to the subject. In some embodiments, the level of TPP1 expression in the vitreous humor and / or aqueous humor was undetectable before administering the rAAV. In some embodiments, the level of TPP1 expression in the subject's serum remains undetectable.

[0020] In some embodiments, the subject is concurrently receiving intraventricular Brineura® enzyme replacement therapy.

[0021] In some embodiments, the subject is a human. [Brief explanation of the drawings]

[0022] 3. Brief description of the drawings [Figure 1A] Schematic representation of the AAV.CB7.CI.hTPP1co.RBG vector genome. ITR represents AAV2 inverted terminal repeats. CB7 represents chicken beta-actin promoter with cytomegalovirus enhancer. RBG polyA represents rabbit beta-globin polyadenylation signal.

[0023] [Figure 1B] 1 provides a map of the production plasmid for the AAV.hTPP1co vector.

[0024] [Figure 1C]A map of the AAV cis-plasmid construct is provided. ITR: inverted terminal repeat, CMV IE promoter: cytomegalovirus immediate early promoter, CB promoter: chicken β-actin promoter chicken β-actin intron, hCLN2: human CLN2 cDNA, rabbit globin polyA: rabbit beta globin polyadenylation signal, Kan-r: kanamycin resistance gene.

[0025] [Figure 1D] 1 provides a map of the AAV trans-packaging plasmid construct.

[0026] [Figure 1E] 1 provides a map of the adenovirus helper plasmid.

[0027] [Figure 2] Provides the CLN2 CRS-MX scoring flowchart.

[0028] [Figure 3] Provides a CLN2 CRS-LX scoring flowchart for subjects under 2-3 years of age.

[0029] [Figure 4A] Figure 1 shows TPP1 concentrations in both aqueous and vitreous humor of cynomolgus monkeys after subretinal injection of Construct I. Dose is GC / eye and data are presented as mean + / - SEM. [Figure 4B] Figure 1 shows TPP1 concentrations in both aqueous and vitreous humor of cynomolgus monkeys after subretinal injection of Construct I. Dose is GC / eye and data are presented as mean + / - SEM.

[0030] [Figure 5] 1 shows serum TPP1 concentrations in cynomolgus monkeys after subretinal injection of Construct I. Dose is GC / eye and data are presented as mean + / - SEM.

[0031] [Figure 6A] Figure 1 shows vector DNA detected in the eye after subretinal injection of Construct I into cynomolgus monkeys. Data are presented as mean and standard deviation. [Figure 6B] Figure 1 shows vector DNA detected in the eye after subretinal injection of Construct I into cynomolgus monkeys. Data are presented as mean and standard deviation.

[0032] [Figure 7A] Figure 1 shows the peripheral biodistribution of Construct I vector DNA 4 weeks after subretinal injection. Below the limit of quantitation (BLQ): 50 copies / μg of DNA. Data are presented as mean and standard deviation. [Figure 7B] Figure 1 shows the peripheral biodistribution of Construct I vector DNA after 13 weeks of subretinal injection. Below the limit of quantitation (BLQ): 50 copies / μg of DNA. Data are presented as mean and standard deviation.

[0033] [Figure 8] Immunofluorescent staining of TPP1 in cynomolgus monkey retina after administration of Construct I. Strips of immunofluorescent staining for TPP1, Nissl counterstain showing retinal cell layers.

[0034] [Figure 9] Threshold image analysis of TPP1 immunoreactivity. Data shown are compared to vehicle-injected animals; a subretinal dose of 1 x 10 GC / eye resulted in a significantly greater area of ​​TPP1 immunostaining (**p=0.0013 vs. either vehicle, one-way ANOVA, post hoc Bonferroni), indicating higher transduction levels at this higher dose.

[0035] [Figure 10]Transduction of CLN2 patient RPE with RGX-381. CLN2 RPE demonstrates the absence of TPP1 expression compared to control RPE. CLN2 RPE transduced with three different doses express TPP1 in a dose-dependent manner after 21 days of treatment.

[0036] [Figure 11] Characterization of retinal organoids. Retinal organoids were characterized at days 80 and 200 of differentiation. TPP1 and recoverin were expressed at day 200, but not at day 80 (Figure 11A). In the absence of TPP1, SCMAS accumulation in CLN2 RO was detected at day 200 (Figure 11B).

[0037] [Figure 12] Treatment of RO with RGX-381. On day 88, RO treated with RGX-381 prevented the SCMAS accumulation detected in untreated CLN2 RO. MOI 3: Mode of infection was 3E+06 GC / cell. DETAILED DESCRIPTION OF THE INVENTION

[0038] 4. MODE FOR CARRYING OUT THE INVENTION Provided herein are methods and compositions for treating Batten disease, particularly ocular symptoms associated with Batten disease. Ocular symptoms associated with Batten disease include, but are not limited to, vision loss, retinal atrophy, and / or retinal degeneration. Such compositions include recombinant adeno-associated viruses (rAAVs), the rAAVs containing AAV capsids, and vector genomes packaged therein, the vector genomes containing (a) AAV 5' inverted terminal repeat (ITR) sequences, (b) a promoter, (c) a CLN2 coding sequence encoding human TPP1, and (d) an AAV 3' ITR as described in Section 4.1. Also provided herein are methods for producing the rAAVs described herein using suspension cell cultures, as described in Section 4.1.8. Compositions comprising recombinant AAVs, such as those described in Section 4.2, are also provided. Also provided herein are methods for treating ocular symptoms of Batten disease caused by a defect in the CLN2 gene, comprising delivering a vector (such as an rAAV) encoding TPP1 to a subject in need thereof, as described herein, e.g., in Section 4.3.

[0039] 4.1 Constructs and Formulations A recombinant viral vector encoding a therapeutic product (e.g., TPP1) for use in the methods provided herein. In some embodiments, the vector is a targeted vector, e.g., a vector that targets retinal pigment epithelial cells.

[0040] In some aspects, the disclosure provides a nucleic acid for use, wherein the nucleic acid encodes a therapeutic product operably linked to a promoter or enhancer-promoter described herein.

[0041] In certain embodiments, provided herein are recombinant vectors comprising one or more nucleic acids (e.g., polynucleotides). The nucleic acids may comprise DNA, RNA, or a combination of DNA and RNA. In certain embodiments, the DNA comprises one or more sequences selected from the group consisting of a promoter sequence, a sequence encoding a therapeutic product of interest, an untranslated region, and a termination sequence. In certain embodiments, the recombinant vectors provided herein comprise a promoter operably linked to a sequence encoding a therapeutic product of interest.

[0042] In certain embodiments, the nucleic acids (e.g., polynucleotides) and nucleic acid sequences disclosed herein can be codon optimized, for example, via any codon optimization technique known to those of skill in the art (see, e.g., review by Quax et al., 2015, Mol Cell 59:149-161).

[0043] 4.1.1 Recombinant viral vectors Recombinant viral vectors include recombinant adenoviruses, adeno-associated viruses (AAV, e.g., AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAVrhlO), lentiviruses, helper-dependent adenoviruses, herpes simplex viruses, poxviruses, Sendai virus (hemagglutinin virus of Japan (HVJ)), alphaviruses, vaccinia viruses, and retroviral vectors. Retroviral vectors include murine leukemia virus (MLV) and human immunodeficiency virus (HIV)-based vectors. Alphavirus vectors include Semliki Forest virus (SFV) and Sindbis virus (SIN). In certain embodiments, the recombinant viral vectors provided herein are modified so that they are replication-deficient in humans. In certain embodiments, the recombinant viral vector is a hybrid vector, e.g., an AAV vector configured in a "disabled" adenoviral vector. In certain embodiments, provided herein is a recombinant viral vector comprising a viral capsid from a first virus and a viral envelope protein from a second virus. In certain embodiments, the second virus is vesicular stomatitis virus (VSV). In more specific embodiments, the envelope protein is VSV-G protein.

[0044] In certain embodiments, the recombinant viral vectors provided herein are AAV-based viral vectors. In a preferred embodiment, the recombinant viral vectors provided herein are AAV9-based viral vectors. In certain embodiments, the AAV9-based viral vectors provided herein retain tropism for retinal cells. In certain embodiments, the AAV-based vectors provided herein encode the AAV rep gene (required for replication) and / or the AAV cap gene (required for capsid protein synthesis). Multiple AAV serotypes have been identified. In certain embodiments, the AAV-based vectors provided herein comprise components from one or more serotypes of AAV. In certain embodiments, the AAV-based vectors provided herein comprise capsid components from one or more of AAV1, AAV2, AAV2tYF, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, or AAVrhlO. In preferred embodiments, the AAV-based vectors provided herein comprise components from one or more of the AAV8, AAV9, AAV10, AAV11, or AAVrhlO serotypes.

[0045] In specific embodiments, an AAV9 vector is provided that includes a viral genome containing a cassette for expression of a therapeutic product under the control of regulatory elements and flanked by ITRs, and a viral capsid having the amino acid sequence of an AAV9 capsid protein or at least 95%, 96%, 97%, 98%, 99%, or 99.9% identical to the amino acid sequence of the AAV9 capsid protein (SEQ ID NO: 9) while retaining the biological function of the AAV9 capsid. In certain embodiments, the encoded AAV9 capsid has the sequence of SEQ ID NO: 9 with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid substitutions, thereby retaining the biological function of the AAV9 capsid.

[0046] AAV9-based viral vectors are used in certain embodiments of the methods described herein. The nucleic acid sequences of AAV-based viral vectors, as well as methods for producing recombinant AAV and AAV capsids, are described, for example, in U.S. Patent No. 7,282,199 B2, U.S. Patent No. 7,790,449 B2, U.S. Patent No. 8,318,480 B2, U.S. Patent No. 8,962,332 B2, and International Application No. PCT / EP2014 / 076466 (each of which is incorporated herein by reference in its entirety). In one aspect, provided herein is an AAV (e.g., AAV9)-based viral vector encoding a therapeutic product.

[0047] In certain embodiments, single-stranded AAV (ssAAV) can be used above.In certain embodiments, self-complementary vectors, such as scAAV, can be used (see, for example, Wu, 2007, Human Gene Therapy, 18(2):171-82; McCarty et al, 2001, Gene Therapy, Vol. 8, Number 16, Pages 1248-1254; and U.S. Patent Nos. 6,596,535; 7,125,717; and 7,456,683 (each of which is incorporated herein by reference in its entirety)).

[0048] In certain embodiments, the recombinant viral vector used in the methods described herein is a recombinant adenoviral vector. The recombinant adenovirus can be a first-generation vector with an E1 deletion, with or without an E3 deletion, and an expression cassette inserted into either deleted region. The recombinant adenovirus can be a second-generation vector containing a complete or partial deletion of the E2 and E4 regions. Helper-dependent adenoviruses retain only the adenoviral inverted terminal repeats and packaging signal (phi). The therapeutic product is inserted between the packaging signal and the 3' ITR, with or without a stuffer sequence to maintain the genome near the wild-type size of approximately 36 kb. Exemplary protocols for producing adenoviral vectors can be found in Alba et al., 2005, "Gutless adenovirus: last generation adenovirus for gene therapy," Gene Therapy 12:S18-S27 (incorporated herein by reference in its entirety).

[0049] 4.1.2 Promoters and Modifiers of Gene Expression In certain embodiments, the recombinant vectors provided herein comprise components that regulate delivery or expression of a therapeutic product (e.g., "expression control elements"). In certain embodiments, the recombinant vectors provided herein comprise components that regulate expression of a therapeutic product. In certain embodiments, the recombinant vectors provided herein comprise components that affect binding or targeting to a cell. In certain embodiments, the recombinant vectors provided herein comprise components that affect localization of a polynucleotide encoding a therapeutic product within a cell after uptake. In certain embodiments, the recombinant vectors provided herein comprise components that can be used, for example, as a detectable or selectable marker to detect or select cells that have uptaken a polynucleotide encoding a therapeutic product.

[0050] In certain embodiments, the recombinant vectors provided herein comprise one or more promoters. In certain embodiments, the promoter is a constitutive promoter. In certain embodiments, the promoter is an inducible promoter. Inducible promoters may be preferred so that the expression of the therapeutic product can be turned on and off as desired for therapeutic efficacy. Such promoters include, for example, hypoxia-inducible promoters and drug-inducible promoters, such as promoters induced by rapamycin and related agents. Hypoxia-inducible promoters include promoters with HIF binding sites; see, for example, Schodel, et al., 2011, Blood 117(23):e207-e217 and Kenneth and Rocha, 2008, Biochem J. 414:19-29 (each incorporated by reference for teachings on hypoxia-inducible promoters). Additionally, hypoxia-inducible promoters that can be used in the constructs include the erythropoietin promoter and the N-WASP promoter (see Tsuchiya, 1993, J. Biochem. 113:395 for a disclosure of the erythropoietin promoter and Salvi, 2017, Biochemistry and Biophysics Reports 9:13-21 for a disclosure of the N-WASP promoter, both of which are incorporated by reference for their teachings on hypoxia-inducible promoters). Alternatively, the recombinant vector may contain a drug-inducible promoter, such as a promoter inducible by administration of rapamycin and related analogues (see, e.g., International Patent Application Publication Nos. WO 94 / 18317, WO 96 / 20951, WO 96 / 41865, WO 99 / 10508, WO 99 / 10510, WO 99 / 36553, and WO 99 / 41258, and U.S. Pat. No. 7,067,526 (disclosing rapamycin analogues), which are incorporated herein by reference for their disclosure of drug-inducible promoters).Inducible promoters can also be selected from known promoters, including ecdysone promoters, estrogen-responsive promoters, and tetracycline-responsive promoters, or heterodimeric repressor switches. See Sochor et al., An Autogenously Regulated Expression System for Gene Therapeutic Ocular Applications. Scientific Reports, 2015 Nov 24; 5: 17105 and Daber R, Lewis M., A novel molecular switch. J Mol Biol. 2009 Aug 28; 391(4): 661-70, Epub 2009 Jun 21 (both of which are incorporated herein by reference in their entirety).

[0051] In certain embodiments, the promoter is a hypoxia-inducible promoter. In certain embodiments, the promoter comprises a hypoxia-inducible factor (HIF) binding site. In certain embodiments, the promoter comprises a HIF-1α binding site. In certain embodiments, the promoter comprises a HIF-2α binding site. In certain embodiments, the HIF binding site comprises an RCGTG motif. For details regarding the location and sequence of HIF binding sites, see, e.g., Schodel, et al., Blood, 2011, 117(23):e207-e217 (incorporated herein by reference in its entirety). In certain embodiments, the promoter comprises a binding site for a hypoxia-inducible transcription factor other than a HIF transcription factor. In certain embodiments, the recombinant vectors provided herein comprise one or more IRES sites that are preferentially translated under hypoxic conditions. For teachings regarding hypoxia-inducible gene expression and factors involved therein, see, e.g., Kenneth and Rocha, Biochem J., 2008, 414:19-29 (incorporated herein by reference in its entirety).

[0052] In another embodiment, the promoter is cell-specific. The term "cell-specific" means that the particular promoter selected for the recombinant vector is capable of directing expression of the optimized TPP1 coding sequence in a particular cell or tissue type.

[0053] In certain embodiments, the promoter is a ubiquitous or constitutive promoter.

[0054] In certain embodiments, the promoter is the CB7 promoter (see Dinculescu et al., 2005, Hum Gene Ther 16:649-663, incorporated herein by reference in its entirety). In certain embodiments, the CB7 promoter includes other expression control elements that enhance expression of the therapeutic product driven by the vector, such as (1) the CAG promoter; (2) the CBA promoter; (3) the CMV promoter; (4) the 1.7 kb red cone opsin promoter (PR1.7 promoter); (5) the rhodopsin kinase (GRK1) photoreceptor-specific enhancer-promoter (Young et al., 2003, Retinal Cell Biology; 44:4076-4085); (6) the hCARp promoter, which is the human cone arrestin promoter; (7) hRKp, which is the rhodopsin kinase promoter; (8) the cone photoreceptor-specific human arrestin 3 (ARR3) promoter; (9) the rhodopsin promoter; and (10) the U6 promoter (particularly when the therapeutic product is a small RNA, e.g., shRNA or siRNA).

[0055] In certain embodiments, the promoter is a hybrid chicken β-actin (CBA) promoter with a cytomegalovirus (CMV) enhancer element, such as the sequence set forth in SEQ ID NO: 5 from nt 3396 to 4061. In another embodiment, the promoter is a CB7 promoter. Other suitable promoters include the human β-actin promoter, the human elongation factor-1α promoter, the cytomegalovirus (CMV) promoter, the simian virus 40 promoter, and the herpes simplex virus thymidine kinase promoter. See, e.g., Damdindorj et al., (August 2014) A Comparative Analysis of Constitutive Promoters Located in Adeno-Associated Viral Vectors. PLoS ONE 9(8):e106472. Still other suitable promoters include viral promoters, constitutive promoters, and regulatable promoters (see, e.g., WO2011 / 126808 and WO2013 / 04943). Alternatively, promoters that respond to physiological cues may be used in the expression cassettes, rAAV genomes, vectors, plasmids, and viruses described herein. In one embodiment, the promoter is small, less than 1000 bp, due to the size limitations of AAV vectors. In another embodiment, the promoter is small, less than 400 bp. Other promoters can be selected by those skilled in the art.

[0056] In further embodiments, the promoter is selected from an SV40 promoter, a dihydrofolate reductase promoter, a phage lambda (PL) promoter, a herpes simplex virus (HSV) promoter, a tetracycline-controlled transactivator-responsive promoter (tet) system, a long terminal repeat (LTR) promoter (e.g., RSV LTR, MoMLV LTR, BIV LTR, or HIV LTR), a Moloney murine sarcoma virus U3 region promoter, a granzyme A promoter, a regulatory sequence(s) of the metallothionein gene, a CD34 promoter, a CD8 promoter, a thymidine kinase (TK) promoter, a B19 parvovirus promoter, a PGK promoter, a glucocorticoid promoter, a heat shock protein (HSP) promoter (e.g., HSP65 and HSP70 promoters), an immunoglobulin promoter, an MMTV promoter, a Rous sarcoma virus (RSV) promoter, a lac promoter, a CaMV 35S promoter, a nopaline synthetase promoter, an MND promoter, or an MNC promoter. The promoter sequences are known to those skilled in the art or are publicly available, eg in the literature or in databases such as GenBank, PubMed, etc.

[0057] In certain embodiments, other expression control elements include a chicken β-actin intron and / or a rabbit β-globin polyA signal. In certain embodiments, the promoter comprises a TATA box. In certain embodiments, the promoter comprises one or more elements. In certain embodiments, one or more promoter elements may be inverted or displaced relative to each other. In certain embodiments, the promoter elements are arranged to function coordinately. In certain embodiments, the promoter elements are arranged to function independently. In certain embodiments, the recombinant vectors provided herein comprise one or more promoters selected from the group consisting of a human CMV immediate early gene promoter, an SV40 early promoter, a Rous sarcoma virus (RS) long terminal repeat, and a rat insulin promoter. In certain embodiments, the recombinant vectors provided herein comprise one or more long terminal repeat (LTR) promoters selected from the group consisting of AAV, MLV, MMTV, SV40, RSV, HIV-1, and HIV-2 LTRs. In certain embodiments, the recombinant vectors provided herein comprise one or more tissue-specific promoters (e.g., retinal pigment epithelial cell-specific promoters). In certain embodiments, the recombinant vectors provided herein comprise an RPE65 promoter. In certain embodiments, the recombinant vectors provided herein comprise a VMD2 promoter.

[0058] In certain embodiments, the recombinant vectors provided herein comprise one or more control elements other than a promoter. In certain embodiments, the recombinant vectors provided herein comprise an enhancer. In certain embodiments, the recombinant vectors provided herein comprise a repressor. In certain embodiments, the recombinant vectors provided herein comprise an intron or a chimeric intron. In certain embodiments, the recombinant vectors provided herein comprise a polyadenylation sequence.

[0059] 4.1.3 Signal peptide In certain embodiments, where the therapeutic product is a therapeutic protein, the recombinant vectors provided herein include components that regulate protein delivery. In certain embodiments, the recombinant vectors provided herein include one or more signal peptides. A signal peptide may also be referred to herein as a "leader sequence" or "leader peptide." In certain embodiments, the signal peptide enables the therapeutic product to achieve proper packaging (e.g., glycosylation) within the cell. In certain embodiments, the signal peptide enables the therapeutic product to achieve proper localization within the cell. In certain embodiments, the signal peptide enables the therapeutic product to achieve secretion from the cell. Examples of signal peptides used in connection with the recombinant vectors and therapeutic products provided herein can be found in Table 1. [Table 1]

[0060] 4.1.4 Untranslated Regions In certain embodiments, where the therapeutic product is a therapeutic protein, the recombinant vectors provided herein comprise one or more untranslated regions (UTRs), e.g., 3' and / or 5' UTRs. In certain embodiments, the UTRs are optimized for a desired protein expression level. In certain embodiments, the UTRs are optimized for the half-life of the mRNA encoding the therapeutic protein. In certain embodiments, the UTRs are optimized for the stability of the mRNA encoding the therapeutic protein. In certain embodiments, the UTRs are optimized for the secondary structure of the mRNA encoding the therapeutic protein.

[0061] 4.1.5 Inverted terminal repeats In certain embodiments, the recombinant viral vectors provided herein comprise one or more inverted terminal repeat (ITR) sequences. The ITR sequences can be used to package a recombinant therapeutic product expression cassette into the virion of the recombinant viral vector. In certain embodiments, the ITRs are derived from AAV, such as AAV8 or AAV2 (see, e.g., Yan et al., 2005, J. Virol., 79(1):364-379; U.S. Patent Nos. 7,282,199 B2, 7,790,449 B2, 8,318,480 B2, 8,962,332 B2, and International Patent Application No. PCT / EP2014 / 076466, each of which is incorporated herein by reference in its entirety).

[0062] 4.1.6 Therapeutic Products In another aspect, provided herein are methods of delivering a therapeutic product to a human subject. In some embodiments, the therapeutic product is TPP1. In some embodiments, the patient is experiencing ocular symptoms associated with Batten disease, including, but not limited to, vision loss, retinal atrophy, and / or retinal degeneration. In certain embodiments, provided herein are methods of delivering an effective amount of a TPP1 therapeutic product to a subject, wherein the subject is experiencing ocular symptoms associated with Batten disease.

[0063] In certain embodiments of the methods described herein, the therapeutic product is tripeptidyl-peptidase 1 (TPP1). The TPP1 gene, also known as CLN2, encodes tripeptidyl-peptidase 1, a lysosomal serine protease with tripeptidyl-peptidase I activity. It is also believed to generate tripeptidyl-peptidase 1 from degradation products produced by lysosomal proteinases and act as a nonspecific lysosomal peptidase requiring substrates with unsubstituted N-termini. As used herein, the terms "TPP1," "CLN2," and "tripeptidyl-peptidase 1" are used interchangeably when referring to the coding sequence. The native nucleic acid sequence encoding human tripeptidyl-peptidase 1 is reported in NCBI Reference Sequence NM_000391.3 and is reproduced herein as SEQ ID NO:2. Two isoforms of human tripeptidyl-peptidase 1 are reported as UniProtKB / Swiss-Prot accessions O14773-1 and O14773-2 (reproduced here as SEQ ID NOs: 1 and 4). Mutations in the CLN2 gene are associated with late infantile NCL (LINCL) disease.

[0064] In certain embodiments, an optimized cDNA encoding human (h)TPP1 may be custom designed and synthesized for optimal codon usage. In certain embodiments, the hTPP1 cDNA, reproduced as SEQ ID NO:3, may then be placed into a transgene expression cassette driven by the CB7 promoter, a hybrid between the cytomegalovirus (CMV) immediate-early enhancer (C4) and the chicken beta-actin promoter; transcription from this promoter is enhanced by the presence of the chicken beta-actin intron (CI) (Figures 1A and 1B). In certain embodiments, the polyA signal for the expression cassette is rabbit beta-globin (RBG) polyA.

[0065] In one aspect, a codon-optimized engineered nucleic acid sequence encoding human (h)TPP1 is provided. In certain embodiments, an engineered human (h)TPP1 cDNA designed to maximize translation compared to the native TPP1 sequence (SEQ ID NO: 2) is provided herein (as SEQ ID NO: 3). Preferably, the codon-optimized TPP1 coding sequence has less than about 80% identity, preferably about 75% identity, to the full-length native TPP1 coding sequence (SEQ ID NO: 2). In one embodiment, the codon-optimized TPP1 coding sequence has about 74% identity to the native TPP1 coding sequence of SEQ ID NO: 2. In one embodiment, the codon-optimized TPP1 coding sequence is characterized by an improved translation rate compared to native TPP1 after AAV-mediated delivery (e.g., rAAV). In one embodiment, the codon-optimized TPP1 coding sequence shares less than about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61% or less identity to the full-length native TPP1 coding sequence of SEQ ID NO: 2. In one embodiment, the codon-optimized nucleic acid sequence is a variant of SEQ ID NO: 3. In another embodiment, the codon-optimized nucleic acid sequence shares about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%, 62%, 61% or more identity with SEQ ID NO: 3. In one embodiment, the codon-optimized nucleic acid sequence is SEQ ID NO: 3. In another embodiment, the nucleic acid sequence is codon optimized for human expression. In other embodiments, a different TPP1 coding sequence is selected.

[0066] The length of sequence identity comparison can be over the entire length of a genome, the entire length of a gene coding sequence, or a fragment of at least about 500 to 5000 nucleotides. However, identity over smaller fragments, e.g., over at least about 9 nucleotides, usually at least about 20 to 24 nucleotides, at least about 28 to 32 nucleotides, or at least about 36 or more nucleotides, can also be desired.

[0067] Percent identity can be readily determined for amino acid sequences spanning the entire length of a protein, polypeptide, about 32 amino acids, about 330 amino acids, or peptide fragments thereof, or the corresponding nucleic acid sequence coding sequence. Suitable amino acid fragments may be at least about 8 amino acids in length and may be up to about 700 amino acids in length. Generally, when referring to "identity," "homology," or "similarity" between two different sequences, the "identity," "homology," or "similarity" is determined with reference to "aligned" sequences. "Aligned" sequences or "alignment" refer to multiple nucleic acid or protein (amino acid) sequences, often including corrections for missing or additional bases or amino acids compared to a reference sequence.

[0068] Identity can be determined by preparing an alignment of sequences and using various algorithms and / or computer programs known in the art or commercially available (e.g., BLAST; ExPASy; ClustalO; FASTA; e.g., using the Needleman-Wunsch algorithm, Smith-Waterman algorithm). Alignment can be performed using any of a variety of publicly or commercially available multiple sequence alignment programs. Sequence alignment programs, such as "Clustal Omega," "Clustal X," "MAP," "PIMA," "MSA," "BLOCKMAKER," "MEME," and "Match-Box," are available for amino acid sequences. Generally, one of these programs is used with default settings, but one of skill in the art can change these settings as needed. Alternatively, one of skill in the art can use another algorithm or computer program that provides at least the same level of identity or alignment as provided by the referenced algorithm and program. See, e.g., JD Thomson et al., Nucl. Acids. Res., "A comprehensive comparison of multiple sequence alignments", 27(13):2682-2690 (1999).

[0069] For nucleic acid sequences, several sequence alignment programs are also available. Examples of such programs include "Clustal Omega," "Clustal W," "CAP sequence assembly," "BLAST," "MAP," and "MEME," which are accessible via web servers on the Internet. Other sources of such programs are known to those skilled in the art. Alternatively, the Vector NTI utility can be used. There are also several algorithms known in the art that can be used to measure nucleotide sequence identity, including those included in the above programs. As another example, polynucleotide sequences can be compared using Fasta™, a program in GCG version 6.1. Fasta™ provides alignments and percent sequence identity of the optimal overlap regions between the query and search sequences. For example, percent sequence identity between nucleic acid sequences can be determined using Fasta™ with its default parameters (word size 6 and NOPAM factor in the scoring matrix) as provided in GCG version 6.1, which is incorporated herein by reference.

[0070] Codon-optimized coding regions can be designed by a variety of different methods. This optimization can be performed using methods available online (e.g., GeneArt), published methods, or companies that provide codon optimization services, such as DNA2.0 (Menlo Park, CA). One codon optimization method is described, for example, in U.S. International Patent Publication No. 2015 / 012924, which is incorporated by reference in its entirety. See also, for example, U.S. Patent Publication Nos. 2014 / 0032186 and 2006 / 0136184. Preferably, the entire length of the product open reading frame (ORF) is modified. However, in some embodiments, only a fragment of the ORF may be altered. Using one of these methods, frequencies can be applied to any given polypeptide sequence to generate a nucleic acid fragment of a codon-optimized coding region that encodes the polypeptide.

[0071] Several options are available for making the actual codon changes or synthesizing the codon-optimized coding region designed as described herein. Such modifications or synthesis can be performed using standard and routine molecular biological procedures well known to those skilled in the art. In one approach, a series of complementary oligonucleotide pairs, each 80-90 nucleotides in length and spanning the length of the desired sequence, are synthesized by standard methods. These oligonucleotide pairs are synthesized such that, upon annealing, they form 80-90 base pair double-stranded fragments containing sticky ends; for example, each oligonucleotide in the pair is synthesized to extend 3, 4, 5, 6, 7, 8, 9, 10, or more bases beyond the region complementary to the other oligonucleotide in the pair. The single-stranded end of each oligonucleotide pair is designed to anneal to the single-stranded end of another oligonucleotide pair. The oligonucleotide pairs are annealed, and then approximately five to six of these double-stranded fragments are annealed together via sticky single-stranded ends, then ligated together, and cloned into a standard bacterial cloning vector, such as the TOPO® vector available from Invitrogen Corporation (Carlsbad, Calif.). The construct is then sequenced by standard methods. Several of these constructs are prepared, consisting of five to six 80-90 base pair fragments ligated together, i.e., approximately 500 base pair fragments, so that the entire desired sequence is represented in a series of plasmid constructs. The inserts of these plasmids are then cleaved with appropriate restriction enzymes and ligated together to form the final construct. The final construct is then cloned into a standard bacterial cloning vector and sequenced. Additional methods will be readily apparent to those skilled in the art. In addition, gene synthesis is readily available commercially.

[0072] In one embodiment, the nucleic acid sequence encoding TPP1 further comprises a nucleic acid encoding a tag polypeptide covalently linked thereto. The tag polypeptide may be selected from known "epitope tags," including, but not limited to, myc tag polypeptide, glutathione-S-transferase tag polypeptide, green fluorescent protein tag polypeptide, myc-pyruvate kinase tag polypeptide, His6 tag polypeptide, influenza virus hemagglutinin tag polypeptide, Flag tag polypeptide, and maltose binding protein tag polypeptide.

[0073] In another aspect, an expression cassette is provided comprising a nucleic acid sequence encoding TPP1. In one embodiment, the sequence is a codon-optimized sequence. In another embodiment, the codon-optimized nucleic acid sequence is SEQ ID NO: 3, which encodes human TPP1.

[0074] In some embodiments, the nucleotide sequence encoding TPP1 may be operably linked to regulatory sequences (including but not limited to initiator sequences, enhancer sequences, and promoter sequences) that induce, repress, or otherwise control transcription of the protein encoding nucleic acid sequence to which they are operably linked. In some embodiments, the nucleotide sequence encoding TPP1 is a nucleotide sequence described in International Patent Application Publication No. WO 2020 / 102369, which is incorporated herein by reference in its entirety.

[0075] 4.1.7 Constructs In certain embodiments of the methods described herein, the recombinant vectors provided herein comprise the following elements, in the following order: a) a constitutive or hypoxia-inducible promoter sequence, and b) a sequence encoding a therapeutic product. In certain embodiments, the sequence encoding a therapeutic product comprises multiple ORFs separated by IRES elements. In certain embodiments, the sequence encoding a therapeutic product comprises multiple subunits within a single ORF separated by F / F2A sequences.

[0076] In certain embodiments of the methods described herein, the recombinant vectors provided herein comprise the following elements in the following order: a) a first ITR sequence, b) a first linker sequence, c) a constitutive or hypoxia-inducible promoter sequence, d) a second linker sequence, e) an intron sequence, f) a third linker sequence, g) a first UTR sequence, h) a sequence encoding a therapeutic product, i) a second UTR sequence, j) a fourth linker sequence, k) a polyA sequence, l) a fifth linker sequence, and m) a second ITR sequence.

[0077] In certain embodiments, a 6841 bp production plasmid of the AAV.hTPP1co vector (AAV.CB7.CI.hTPP1co.RBG) can be constructed using the hTPP1co expression cassette described herein flanked by AAV2-derived ITRs and resistance to ampicillin as a selectable marker (FIG. 1B). In certain embodiments, a similar AAV.hTPP1co production plasmid with resistance to kanamycin can also be constructed. In certain embodiments, the vector derived from both plasmids can be a single-stranded DNA genome with AAV2-derived ITRs flanking the hTPP1co expression cassette described herein.

[0078] In certain embodiments, the AAV.hTPP1co vector may be produced by triple transfection and formulated with an excipient consisting of phosphate-buffered saline (PBS) containing 0.001% Pluronic F68 (PF68). See, for example, Mizukami, Hiroaki, et al., A Protocol for AAV vector production and purification, Diss. Division of Genetic Therapeutics, Center for Molecular Medicine, 1998. In certain embodiments, the genomic titer of the produced vector may be determined via droplet digital PCR (ddPCR). See, for example, M. Lock et al., Hu Gene Therapy Methods, Hum Gene Ther Methods. 2014 Apr;25(2):115-25. doi:10.1089 / hgtb.2013.131. Epub 2014 Feb 14. An exemplary AAV.hTPP1co vector is described herein, which may be referred to herein as AAV9.CB7.hCLN2.

[0079] In some embodiments, the recombinant vector is a recombinant vector described in International Patent Application Publication No. WO 2020 / 102369, which is incorporated by reference in its entirety.

[0080] As used herein, "expression cassette" refers to a nucleic acid molecule comprising a coding sequence for a TPP1 protein, a promoter, and optionally other regulatory sequences therefor, which cassette may be packaged into the capsid of a viral vector (e.g., a viral particle). Typically, such expression cassettes for generating viral vectors contain the CLN2 sequence described herein, flanked by a packaging signal for the viral genome and other expression control sequences, such as those described herein. For example, in the case of an AAV viral vector, the packaging signal is the 5' inverted terminal repeat (ITR) and the 3' ITR. When packaged into an AAV capsid, the ITRs together with the expression cassette may be referred to herein as the "recombinant AAV (rAAV) genome" or "vector genome." In one embodiment, the expression cassette comprises a codon-optimized nucleic acid sequence encoding a TPP1 protein. In one embodiment, the cassette provides a codon-optimized CLN2 in operative association with expression control sequences that direct expression of the codon-optimized nucleic acid sequence encoding TPP1 in a host cell.

[0081] In another embodiment, an expression cassette for use in an AAV vector is provided. In this embodiment, the AAV expression cassette comprises at least one AAV inverted terminal repeat (ITR) sequence. In another embodiment, the expression cassette comprises a 5' ITR sequence and a 3' ITR sequence. In one embodiment, the 5' and 3' ITRs flank a codon-optimized nucleic acid sequence encoding TPP1, optionally with additional sequences that direct expression of the codon-optimized nucleic acid sequence encoding TPP1 in a host cell. Thus, as described herein, an AAV expression cassette is meant to describe the above expression cassette flanked at its 5' end by a 5' AAV inverted terminal repeat (ITR) and at its 3' end by a 3' AAV ITR. Thus, the rAAV genome comprises the minimal sequences required for packaging the expression cassette into an AAV viral particle, i.e., the AAV 5' and 3' ITRs. The AAV ITRs may be derived from the ITR sequences of any AAV, as described herein. These ITRs may be of the same AAV origin as the capsid used in the resulting recombinant AAV, or may be of a different AAV origin (to produce an AAV pseudotype). In one embodiment, ITR sequences from AAV2 are used. However, ITRs from other AAV sources may also be selected. If the ITR source is from AAV2 and the AAV capsid is from another AAV source, the resulting vector may be referred to as a pseudotype. Typically, an AAV vector genome contains the AAV 5' ITR, the TPP1 coding sequence and any regulatory sequences, and the AAV 3' ITR. However, other configurations of these elements may be suitable. A shortened version of the 5' ITR, called ΔITR, has been described in which the D sequence and terminal separation sites (trs) are deleted. In other embodiments, full-length AAV 5' and 3' ITRs are used. Each rAAV genome can then be introduced into a production plasmid.

[0082] In one aspect, a vector is provided that comprises any of the expression cassettes described herein. As described herein, such vectors can be plasmids of various origins and, in certain embodiments, are useful for generating recombinant replication-defective viruses, as further described herein.

[0083] In one embodiment, the vector is a non-viral plasmid containing the described expression cassette, e.g., "naked DNA," "naked plasmid DNA," RNA, and mRNA, coupled with various compositions and nanoparticles, including, for example, micelles, liposomes, cationic lipid-nucleic acid compositions, polyglycan compositions and other polymers, lipid- and / or cholesterol-based nucleic acid conjugates, and other constructs described herein. See, e.g., X. Su et al., Mol. Pharmaceutics, 2011, 8(3), pp. 774-787; Web publication: March 21, 2011; WO2013 / 182683, WO2010 / 053572, and WO2012 / 170930 (all incorporated herein by reference). Such non-viral TPP1 vectors can be administered by the routes described herein. Viral or non-viral vectors can be formulated with physiologically acceptable carriers for use in gene transfer and gene therapy applications.

[0084] In another embodiment, the vector is a viral vector containing the described expression cassette therein. In some embodiments, the viral vector contains an exogenous or heterologous CLN2 nucleic acid transgene. In one embodiment, the expression cassette described herein can be engineered onto a plasmid used for drug delivery or viral vector production. Suitable viral vectors are preferably replication-deficient and selected from those that target brain cells. Viral vectors can include any virus suitable for gene therapy, including, but not limited to, adenovirus, herpesvirus, lentivirus, retrovirus, parvovirus, etc.

[0085] In some embodiments, the viral vector can be replication-deficient. A "replication-deficient virus" or "viral vector" refers to a synthetic or recombinant viral particle in which an expression cassette containing a gene of interest is packaged within a viral capsid or envelope; any viral genomic sequences packaged within the viral capsid or envelope are replication-deficient, i.e., they are unable to produce progeny virions but retain the ability to infect target cells. In one embodiment, the genome of the viral vector does not contain genes encoding enzymes required for replication (the genome can be engineered to be "gutless," containing only the transgene of interest flanked by signals required for amplification and packaging of the artificial genome), although these genes can be supplied during production. Therefore, replication and infection by progeny virions can only occur in the presence of viral enzymes required for replication, making it considered safe for use in gene therapy.

[0086] In another embodiment, a recombinant adeno-associated virus (rAAV) vector is provided, which compromises the AAV capsid and the vector genome packaged therein.

[0087] In one embodiment, the vector genome comprises (a) an AAV 5' inverted terminal repeat (ITR) sequence, (b) a promoter, (c) a coding sequence encoding human TPP1, and (d) an AAV 3' ITR. In another embodiment, the vector genome is an expression cassette described herein. In one embodiment, the CLN2 sequence encodes the full-length TPP1 protein. In one embodiment, the TPP1 sequence is the protein sequence of SEQ ID NO: 1. In another embodiment, the coding sequence is SEQ ID NO: 3 or a variant thereof.

[0088] ITRs or other AAV components can be readily isolated or engineered from AAV using techniques available to those of skill in the art. Such AAV may be isolated, engineered, or obtained from academic, commercial, or public sources (e.g., American Type Culture Collection, Manassas, VA). Alternatively, AAV sequences may be engineered synthetically or via other suitable means by reference to published sequences, such as those available in the literature or in databases such as GenBank and PubMed. AAV viruses may also be engineered by conventional molecular biology techniques, allowing these particles to be optimized for cell-specific delivery of nucleic acid sequences, to minimize immunogenicity, to tailor stability and particle longevity, for efficient degradation, for precise delivery to the nucleus, etc.

[0089] AAV fragments can be readily utilized in a variety of vector systems and host cells. Desirable AAV fragments include cap proteins, including vp1, vp2, vp3, and hypervariable regions; rep proteins, including rep78, rep68, rep52, and rep40; and sequences encoding these proteins. Such fragments may be used alone, in combination with other AAV serotype sequences or fragments, or in combination with elements derived from other AAV or non-AAV viral sequences. As used herein, artificial AAV serotypes include, but are not limited to, AAVs with non-naturally occurring capsid proteins. Such artificial capsids can be generated by any suitable technique by combining novel AAV sequences of the invention (e.g., fragments of the vp1 capsid protein) with heterologous sequences that can be obtained from another AAV serotype (known or novel), non-contiguous portions of the same AAV serotype, non-AAV viral sources, or non-viral sources. The artificial AAV serotype can be, but is not limited to, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid. In one embodiment, the vector contains AAV9 cap and / or rep sequences. See U.S. Patent No. 7,906,111 (incorporated herein by reference).

[0090] In one embodiment, provided herein is an AAV vector having an AAV9 capsid characterized by the amino acid sequence of SEQ ID NO:6, in which a nucleic acid encoding the classical late infantile neuronal ceroid lipofuscinosis 2 (CLN2) gene is provided under the control of regulatory sequences that direct its expression in a patient in need thereof.

[0091] As used herein, "AAV9 capsid" is characterized as a DNAse-resistant particle that is an assembly of approximately 60 variable proteins (vp) typically expressed as alternative splice variants resulting in different length proteins of SEQ ID NO:6. See also Genbank Accession No. AAS99264.1 (incorporated herein by reference). See also US7906111 and WO2005 / 033321. As used herein, "AAV9 variants" include, for example, those described in WO2016 / 049230, US8,927,514, US2015 / 0344911, and US8,734,809. The amino acid sequence is reproduced in SEQ ID NO:6, and the coding sequence is reproduced in SEQ ID NO:7. In one embodiment, the AAV9 capsid comprises the capsid encoded by SEQ ID NO: 7, or a sequence sharing at least about 90%, 95%, 95%, 98% or 99% identity therewith.

[0092] The largest protein, vp1, generally spans the entire amino acid sequence of SEQ ID NO:6 (aa1-736 of SEQ ID NO:6). In certain embodiments, the AAV9 vp2 protein has the amino acid sequence of SEQ ID NO:6 from 138 to 736. In certain embodiments, the AAV9 vp3 has the amino acid sequence of SEQ ID NO:6 from 203 to 736. In certain embodiments, the vp1, 2, or 3 proteins can be truncated (e.g., one or more amino acids at the N- or C-terminus). The AAV9 capsid is composed of approximately 60 vp proteins, with vp1, vp2, and vp3 present in the assembled capsid in a ratio of about 1 vp to about 1 vp2 to about 10-20 vp3 proteins. This ratio may vary depending on the production system used. In certain embodiments, engineered AAV9 capsids can be produced in the absence of vp2.

[0093] Designing a nucleic acid sequence encoding this AAV9 capsid, including DNA (genomic or cDNA) or RNA (e.g., mRNA), is within the skill in the art. In certain embodiments, a nucleic acid sequence encoding the AAV9 vp1 capsid protein is provided in SEQ ID NO:7. In other embodiments, a nucleic acid sequence 70% to 99.9% identical to SEQ ID NO:7 may be selected to express the AAV9 capsid. In certain other embodiments, the nucleic acid sequence is at least about 75% identical, at least 80% identical, at least 85%, at least 90%, at least 95%, at least 97% identical, or at least 99% to 99.9% identical to SEQ ID NO:7.

[0094] As used herein, the term "clade" as it relates to a group of AAVs refers to a group of AAVs that are systematically related to each other, as determined using a neighbor-joining algorithm based on an alignment of AAV vp1 amino acid sequences with a bootstrap value of at least 75% (out of at least 1,000 replicates) and a Poisson-corrected distance measure of 0.05 or less. Neighbor-joining algorithms have been described in the literature. See, for example, M. Nei and S. Kumar, Molecular Evolution and Phylogenetics, Oxford University Press, New York (2000). Computer programs that can be used to implement this algorithm are available. For example, the MEGA v2.1 program implements a modified Nei-Gojobori method. Using these techniques and computer programs, as well as the sequence of the AAV vp1 capsid protein, one skilled in the art can easily determine whether a selected AAV is included in one of the clades identified herein, another clade, or outside these clades. See, e.g., G Gao, et al, J Virol, 2004 Jun;78(10):6381-6388 (identifying clades A, B, C, D, E, and F and providing nucleic acid sequences of novel AAVs, GenBank accession numbers AY530553 to AY530629). See also WO2005 / 033321. AAV9 is further characterized as being within clade F. Other clade F AAVs include AAVhu31 and AAVhu32.

[0095] As used herein, the term variant, in the context of AAV, refers to any AAV sequence derived from a known AAV sequence, including those that share at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 99%, or more amino acid or nucleic acid sequence identity. In another embodiment, the AAV capsid includes variants that may contain up to about 10% variation from a described or known AAV capsid sequence. That is, the AAV capsid shares about 90% identity to about 99.9% identity, about 95% to about 99% identity, or about 97% to about 98% identity with an AAV capsid provided herein and / or known in the art. In one embodiment, the AAV capsid shares at least 95% identity with an AAV9 capsid. When determining percent identity of an AAV capsid, comparison can be made to any of the variable proteins (e.g., vp1, vp2, or vp3). In one embodiment, the AAV capsid shares at least 95% identity with AAV9 over vp1, vp2, or vp3.

[0096] As used herein, "artificial AAV" refers to, but is not limited to, an AAV having a non-naturally occurring capsid protein. Such an artificial capsid can be generated by any suitable technique by combining a selected AAV sequence (e.g., a fragment of the vp1 capsid protein) with a different selected AAV, non-contiguous portions of the same AAV, a non-AAV viral source, or a heterologous sequence that can be obtained from a non-viral source. The artificial AAV can be, but is not limited to, a pseudotyped AAV, a chimeric AAV capsid, a recombinant AAV capsid, or a "humanized" AAV capsid. Pseudotype vectors, in which the capsid of one AAV is replaced with a heterologous capsid protein, are useful in the present invention. In one embodiment, AAV2 / 9 and AAV2 / rh.10 are exemplary pseudotype vectors.

[0097] In another embodiment, a self-complementary AAV is used. The term "self-complementary AAV" refers to a plasmid or vector containing an expression cassette designed so that the coding region carried by the recombinant AAV nucleic acid sequence forms an intramolecular double-stranded DNA template. Upon infection, rather than waiting for cell-mediated synthesis of the second strand, the two complementary halves of the scAAV associate to form a single double-stranded DNA (dsDNA) unit ready for immediate replication and transcription. See, for example, DM McCarty et al., "Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis," Gene Therapy, (August 2001), Vol. 8, Number 16, Pages 1248-1254. Self-complementary AAVs are described, for example, in U.S. Patent Nos. 6,596,535, 7,125,717, and 7,456,683, each of which is incorporated by reference in its entirety.

[0098] In yet another embodiment, an expression cassette comprising any of those described herein is used to generate a recombinant AAV genome.

[0099] In one embodiment, the expression cassettes described herein are engineered into suitable genetic elements (vectors) useful for generating viral vectors and / or for delivery into host cells, e.g., naked DNA, phage, transposons, cosmids, episomes, etc., which transfer the CLN2 sequences carried thereon. The selected vector can be delivered by any suitable method, including transfection, electroporation, liposome delivery, membrane fusion techniques, high-speed DNA-coated pellets, viral infection, and protoplast fusion. Methods used to generate such constructs are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY.

[0100] The ITRs are the only AAV components required in cis in the same construct as the expression cassette for packaging of the expression cassette or rAAV genome or production plasmid into virions. In one embodiment, to generate AAV vectors, the replication (rep) and / or capsid (cap) coding sequences are removed from the AAV genome and supplied in trans or by a packaging cell line.

[0101] Methods for producing and isolating AAV viral vectors suitable for delivery to a subject are known in the art. See, for example, U.S. Patent No. 7,790,449, U.S. Patent No. 7,282,199, WO 2003 / 042397, WO 2005 / 033321, WO 2006 / 110689, and U.S. Patent No. 7,588,772 B2. In one system, a producer cell line is transiently transfected with a construct encoding a transgene flanked by ITRs and a construct(s) encoding rep and cap. In a second system, a packaging cell line that stably supplies rep and cap is transiently transfected with a construct encoding a transgene flanked by ITRs. In certain embodiments, the producer or packaging cell line is a suspension cell line such that the AAV viral vectors described herein can be produced by growing the producer or packaging cell line in suspension culture. In each of these systems, AAV virions are produced in response to infection with a helper adenovirus or herpesvirus, requiring the separation of rAAV from contaminating viruses. More recently, systems have been developed that do not require infection with a helper virus to restore AAV, and the necessary helper functions (i.e., adenovirus E1, E2a, VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29, and herpesvirus polymerase) are also supplied in trans by the system. In these newer systems, helper functions can be supplied by transient transfection of cells with constructs encoding the necessary helper functions, or cells can be engineered to stably contain genes encoding the helper functions, the expression of which can be controlled at the transcriptional or post-transcriptional level.

[0102] In yet another system, an expression cassette flanked by ITRs and rep / cap genes is introduced into insect cells by infection with a baculovirus-based vector. For a review of these production systems generally, see, e.g., Zhang et al., 2009, "Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production," Human Gene Therapy 20:922-929, the contents of which are incorporated herein by reference in their entirety. Methods of making and using these and other AAV production systems are also described in the following U.S. patents, the contents of each of which are incorporated herein by reference in their entirety: 5,139,941, 5,741,683, 6,057,152, 6,204,059, 6,268,213, 6,491,907, 6,660,514, 6,951,753, 7,094,604, 7,172,893, 7,201,898, 7,229,823, and 7,439,065. See generally, e.g., Grieger & Samulski, 2005, "Adeno-associated virus as a gene therapy vector: Vector development, production and clinical applications," Adv. Biochem. Engin / Biotechnol. 99:119-145; Buning et al., 2008, "Recent developments in adeno-associated virus vector technology," J. Gene Med. 10:717-733; and the references cited below, each of which is incorporated herein by reference in its entirety.

[0103] In one aspect, provided herein is a method of producing the rAAV described herein, comprising growing in suspension culture a suspension cell line capable of producing rAAV.

[0104] In certain embodiments, the suspension cell line is derived from an adherent cell line by adapting the cells to suspension culture using serum-free and animal-component-free culture medium, hi certain embodiments, the suspension cell line is a HEK293 suspension cell line.

[0105] The methods used to construct any embodiment of the present invention are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, e.g., Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY (2012). Similarly, methods for producing rAAV virions are well known, and the selection of a suitable method is not a limitation on the present invention. See, e.g., K. Fisher et al., (1993) J. Virol., 70:520-532 and U.S. Patent No. 5,478,745.

[0106] "Plasmids" are generally designated herein by a lower case p preceded and / or followed by capital letters and / or numbers, in accordance with standard naming conventions familiar to those of skill in the art. Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known and readily available to those of skill in the art. Moreover, one of skill in the art can readily construct any number of other plasmids suitable for use in the present invention. The properties, construction, and use of such plasmids, as well as other vectors, in the present invention will be readily apparent to those of skill in the art from the present disclosure.

[0107] In one embodiment, the production plasmid is one described herein or in WO2012 / 158757 (incorporated herein by reference). Various plasmids are known in the art for use in producing rAAV vectors and are useful herein. The production plasmid is cultured in a host cell that expresses AAV cap and / or rep proteins. In the host cell, each rAAV genome is rescued and packaged into capsid or envelope proteins to form infectious viral particles.

[0108] In one aspect, a production plasmid containing the expression cassette described above is provided. In one embodiment, the production plasmid is shown in Figure 1B. This plasmid is used in the example of generating an rAAV-human codon-optimized TPP1 vector. Such a plasmid contains a 5' AAV ITR sequence, a selected promoter, a polyA sequence, and a 3' ITR, and also contains an intron sequence, such as the chicken beta-actin intron. An exemplary schematic is shown in Figure 1A. In further embodiments, the intron sequence maintains the size of the rAAV vector genome between about 3 kilobases (kb) and about 6 kb, between about 4.7 kb and about 6 kb, between about 3 kb and about 5.5 kb, or between about 4.7 kb and 5.5 kb. An example of a production plasmid containing a TPP1 coding sequence can be found in SEQ ID NO: 5. In another embodiment, the production plasmid is modified to optimize vector plasmid production efficiency. Such modifications include the addition of other neutral sequences or the inclusion of a lambda stuffer sequence to adjust the level of supercoiling of the vector plasmid. Such modifications are contemplated herein. In other embodiments, terminators and other sequences are included in the plasmid.

[0109] In certain embodiments, the rAAV expression cassette, vector (such as an rAAV vector), virus (such as an rAAV), and / or production plasmid comprises an AAV inverted terminal repeat, a codon-optimized nucleic acid sequence encoding TPP1, and expression control sequences that direct expression of the encoded protein in a host cell. In other embodiments, the rAAV expression cassette, virus, vector (such as an rAAV vector), and / or production plasmid further comprises one or more of an intron, a Kozak sequence, a polyA, a post-transcriptional regulatory element, etc. In one embodiment, the post-transcriptional regulatory element is a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE).

[0110] Expression cassettes, vectors and plasmids contain other components that can be optimized for a particular species using techniques known in the art, including, for example, codon optimization as described herein.

[0111] In other embodiments, the expression cassettes, vectors, plasmids, and viruses described herein contain other appropriate transcription initiation, termination, and enhancer sequences, efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; TATA sequences; sequences that stabilize cytoplasmic mRNA; sequences that improve translation efficiency (i.e., Kozak consensus sequences); introns; sequences that improve protein stability; and, optionally, sequences that improve secretion of the encoded product. An expression cassette or vector can contain none, one, or more of the elements described herein.

[0112] Examples of suitable poly A sequences include, for example, synthetic poly A or those derived from bovine growth hormone (bGH), human growth hormone (hGH), SV40, rabbit β-globin (RGB), or modified RGB (mRGB). In a further embodiment, the poly A has the nucleic acid sequence of nt 33 to 159 of SEQ ID NO:5.

[0113] Examples of suitable enhancers include, for example, a CMV enhancer, an RSV enhancer, an alpha-fetoprotein enhancer, a TTR minimal promoter / enhancer, an LSP (TH-binding globulin promoter / alpha 1-microglobulin / bikunin enhancer), an APB enhancer, an ABPS enhancer, an alpha mic / bik enhancer, a TTR enhancer, en34, and an ApoE, among others.

[0114] In one embodiment, a Kozak sequence is included upstream of the TPP1 coding sequence to enhance translation from the correct start codon. In another embodiment, a CBA exon 1 and intron are included in the expression cassette. In one embodiment, the TPP1 coding sequence is placed under the control of a hybrid chicken beta-actin (CBA) promoter. This promoter consists of a cytomegalovirus (CMV) immediate-early enhancer, the proximal chicken beta-actin promoter, and CBA exon 1 adjacent to intron 1 sequence.

[0115] In another embodiment, the intron is selected from CBA, human beta globin, IVS2, SV40, bGH, alpha-globin, beta-globin, collagen, ovalbumin, p53, or a fragment thereof.

[0116] In one embodiment, the expression cassettes, vectors, plasmids, and viruses contain a 5' ITR, a chicken beta actin (CBA) promoter, a CMV enhancer, a CBA exon 1 and intron, a human codon-optimized CLN2 sequence, a rabbit globin polyA, and a 3' ITR. In a further embodiment, the expression cassette comprises nt 1 to 4020 of SEQ ID NO:8. In yet another embodiment, the 5' ITR has the nucleic acid sequence from nt 3199 to nt 3328 of SEQ ID NO:5, and the 3' ITR has the nucleic acid sequence from nt 248 to nt 377 of SEQ ID NO:5. In a further embodiment, the production plasmid has the sequence of SEQ ID NO:5, which is also shown in Figures 1C-1E.

[0117] 4.1.8 Vector Production and Testing The recombinant vectors provided herein (e.g., recombinant viral vectors) can be produced using host cells. The recombinant vectors provided herein can be produced using mammalian host cells, such as A549, WEHI, 10T1 / 2, BHK, MDCK, COS1, COS7, BSC1, BSC40, BMT10, VERO, W138, HeLa, 293, Saos, C2C12, L, HT1080, HepG2, primary fibroblasts, hepatocytes, and myoblasts. The recombinant vectors provided herein can be produced using host cells from humans, monkeys, mice, rats, rabbits, or hamsters. In other embodiments, the term "host cells" is intended to refer to brain cells of a subject being treated in vivo for Batten disease. Such host cells include epithelial cells of the CNS, including the ependyma, the epithelial lining of the ventricular system. Other host cells include neurons, astrocytes, oligoedendrocytes, and microglia.

[0118] For recombinant viral vectors, host cells are stably transformed with sequences encoding the therapeutic product and associated elements (i.e., the vector genome), as well as a means of producing the virus in the host cell, e.g., replication and capsid genes (e.g., the AAV rep and cap genes). For methods of producing recombinant AAV vectors with AAV8 capsids, see Section IV of the Detailed Description of the Invention in U.S. Pat. No. 7,282,199 B2 (incorporated herein by reference in its entirety). The genome copy titer of the vector can be determined, for example, by TAQMAN® analysis. Virions can be recovered, for example, by CsCl2 sedimentation.

[0119] In vitro assays, such as cell culture assays, can be used to measure the expression of therapeutic products from the vectors described herein, thus demonstrating, for example, the efficacy of the vectors. For example, the PER.C6® cell line (Lonza), a cell line derived from human embryonic retinal cells, or retinal pigment epithelial cells, such as the retinal pigment epithelial cell line hTERT RPE-1 (available from ATCC®), can be used to evaluate therapeutic product expression. Once expressed, the characteristics of the expressed therapeutic product can be determined, including determining post-translational modification patterns. In addition, the benefits resulting from post-translational modification of cell-expressed therapeutic products can be determined using assays known in the art.

[0120] 4.2 Composition Compositions are described herein that include a recombinant vector encoding a therapeutic product described herein and a suitable carrier. Suitable carriers (e.g., for suprachoroidal, subretinal, juxtascleral, intravitreal, subconjunctival, and / or intraretinal administration) may be readily selected by one of skill in the art.

[0121] The present disclosure provides a pharmaceutical composition comprising a recombinant adeno-associated virus (AAV), potassium phosphate monobasic, sodium chloride, sodium phosphate dibasic anhydrous, sucrose, and a surfactant.

[0122] In some embodiments, the disclosure provides pharmaceutical compositions comprising a recombinant adeno-associated virus (AAV), an ionic salt excipient or buffering agent, sucrose, and poloxamer 188. In some embodiments, the ionic salt excipient or buffering agent can be one or more components consisting of the group consisting of potassium phosphate monobasic, potassium phosphate, sodium chloride, sodium phosphate dibasic anhydrous, sodium phosphate hexahydrate, sodium phosphate monobasic monohydrate, tromethamine, tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), amino acids, histidine, histidine hydrochloride (histidine-HCl), sodium succinate, sodium citrate, sodium acetate, and (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium sulfate, magnesium sulfate, magnesium chloride hexahydrate, calcium sulfate, potassium chloride, calcium chloride, calcium citrate.

[0123] In certain embodiments, the pharmaceutical composition has an ionic strength of about 60 mM to 115 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 60 mM to 100 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 65 mM to 95 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 70 mM to 90 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 75 mM to 85 mM.

[0124] In certain embodiments, the pharmaceutical composition has an ionic strength of about 30 mM to 100 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 35 mM to 95 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 40 mM to 90 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 45 mM to 85 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 50 mM to 80 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 55 mM to 75 mM. In certain embodiments, the pharmaceutical composition has an ionic strength of about 60 mM to 70 mM.

[0125] In certain embodiments, the pharmaceutical composition has an ionic strength in the range of 60 mM to 115 mM. In certain embodiments, the pharmaceutical composition has an ionic strength in the range of 60 mM to 100 mM. In certain embodiments, the pharmaceutical composition has an ionic strength in the range of 65 mM to 95 mM. In certain embodiments, the pharmaceutical composition has an ionic strength in the range of 70 mM to 90 mM. In certain embodiments, the pharmaceutical composition has an ionic strength in the range of 75 mM to 85 mM.

[0126] In certain embodiments, the pharmaceutical composition has an ionic strength range of 30 mM to 100 mM. In certain embodiments, the pharmaceutical composition has an ionic strength range of 35 mM to 95 mM. In certain embodiments, the pharmaceutical composition has an ionic strength range of 40 mM to 90 mM. In certain embodiments, the pharmaceutical composition has an ionic strength range of 45 mM to 85 mM. In certain embodiments, the pharmaceutical composition has an ionic strength range of 50 mM to 80 mM. In certain embodiments, the pharmaceutical composition has an ionic strength range of 55 mM to 75 mM. In certain embodiments, the pharmaceutical composition has an ionic strength range of 60 mM to 70 mM.

[0127] In certain embodiments, the pharmaceutical composition comprises potassium chloride at a concentration of 0.2 g / L.

[0128] In certain embodiments, the pharmaceutical composition comprises potassium phosphate monobasic at a concentration of 0.2 g / L.

[0129] In certain embodiments, the pharmaceutical composition comprises sodium chloride at a concentration of 5.84 g / L,

[0130] In certain embodiments, the pharmaceutical composition comprises sodium phosphate dibasic anhydrous at a concentration of 1.15 g / L.

[0131] In certain embodiments, the pharmaceutical composition contains sucrose at a concentration of 3% (weight / volume, 30 g / L) to 18% (weight / volume, 180 g / L), hi certain embodiments, the pharmaceutical composition contains sucrose at a concentration of 4% (weight / volume, 40 g / L).

[0132] In certain embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.001% (weight / volume, 0.01 g / L).

[0133] In certain embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.0005% (weight / volume, 0.005 g / L) to 0.05% (weight / volume, 0.5 g / L). In certain embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.001% (weight / volume, 0.01 g / L).

[0134] In some embodiments, the present disclosure provides a pharmaceutical composition comprising a recombinant adeno-associated virus (AAV), an ionic salt excipient or buffer, sucrose, and a surfactant. In some embodiments, the ionic salt excipient or buffering agent can be one or more members consisting of the group consisting of potassium phosphate monobasic, potassium phosphate, sodium chloride, sodium phosphate dibasic anhydrous, sodium phosphate hexahydrate, sodium phosphate monobasic monohydrate, tromethamine, tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), amino acids, histidine, histidine hydrochloride (histidine-HCl), sodium succinate, sodium citrate, sodium acetate, and (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium sulfate, magnesium sulfate, magnesium chloride hexahydrate, calcium sulfate, potassium chloride, calcium chloride, calcium citrate. In some embodiments, the surfactant can be one or more members from the group consisting of poloxamer 188, polysorbate 20, and polysorbate 80.

[0135] In certain embodiments, the pharmaceutical composition comprises polysorbate 20 at a concentration of 0.0005% (weight / volume, 0.05 g / L) to 0.05% (weight / volume, 0.5 g / L).

[0136] In certain embodiments, the pharmaceutical composition comprises polysorbate 80 at a concentration of 0.0005% (weight / volume, 0.05 g / L) to 0.05% (weight / volume, 0.5 g / L).

[0137] In certain embodiments, the pH of the pharmaceutical composition is about 7.4.

[0138] In certain embodiments, the pH of the pharmaceutical composition is about 6.0 to 9.0.

[0139] In certain embodiments, the pH of the pharmaceutical composition is 7.4.

[0140] In certain embodiments, the pH of the pharmaceutical composition is 6.0 to 9.0.

[0141] As used herein, and unless otherwise specified, the term "about" means within plus or minus 10% of a given value or range.

[0142] In certain embodiments, the pharmaceutical composition is in a hydrophobic coated glass vial.

[0143] In certain embodiments, the pharmaceutical composition is in a cycloolefin polymer (COP) vial.

[0144] In certain embodiments, the pharmaceutical composition is in a Daikyo Crystal Zenith® (CZ) vial.

[0145] In certain embodiments, the pharmaceutical composition is in a TopLyo-coated vial.

[0146] In certain embodiments, disclosed herein is a pharmaceutical composition consisting of (a) a recombinant AAV, (b) potassium chloride at a concentration of 0.2 g / L, (c) potassium phosphate monobasic at a concentration of 0.2 g / L, (d) sodium chloride at a concentration of 5.84 g / L, (e) sodium phosphate dibasic anhydrous at a concentration of 1.15 g / L, (f) sucrose at a concentration of 4% weight / volume (40 g / L), (g) poloxamer 188 at a concentration of 0.001% weight / volume (0.01 g / L), and (h) water, wherein the recombinant AAV is AAV9.

[0147] In certain embodiments, the vector genome concentration (VGC) of the pharmaceutical composition is about 3×10 9 GC / mL, approximately 1×10 10 GC / mL, approximately 1.2×10 10 GC / mL, approximately 1.6×10 10 GC / mL, approximately 4×10 10 GC / mL, approximately 6×10 10 GC / mL, approximately 1×10 11 GC / mL, approximately 2×10 11 GC / mL, approximately 2.4×10 11 GC / mL, approximately 2.5×10 11 GC / mL, approximately 3×10 11 GC / mL, approximately 6.2×10 11 GC / mL, approximately 1×10 12 GC / mL, approximately 3×10 12 GC / mL, approximately 2×10 13 GC / mL or approximately 3 x 10 13 GC / mL.

[0148] In certain embodiments, the disclosure provides a pharmaceutical composition or formulation comprising a recombinant adeno-associated virus (AAV), potassium phosphate monobasic, sodium chloride, sodium phosphate dibasic anhydrous, sucrose, and poloxamer 188. In some embodiments, the recombinant AAV comprises components from AAV9. In some embodiments, the AAV comprises an AAV capsid and, packaged therein, a vector genome comprising (a) an AAV 5' inverted terminal repeat (ITR) sequence, (b) a promoter, (c) a CLN2 coding sequence encoding human TPP1, and (d) an AAV 3' ITR.

[0149] In a preferred embodiment, the AAV used to deliver the transgene should have tropism for human retinal cells or photoreceptor cells. Such AAV can include non-replicating recombinant adeno-associated viral vectors, with those having an AAV9 capsid being particularly preferred. In another specific embodiment, the viral vector or other DNA expression construct described herein is Construct I, which comprises the following components: an AAV capsid and a vector genome packaged therein, the vector genome comprising (a) an AAV 5' inverted terminal repeat (ITR) sequence, (b) a promoter, (c) a CLN2 coding sequence encoding human TPP1, and (d) an AAV 3' ITR.

[0150] In some embodiments, the pharmaceutical composition consists of (a) an AAV capsid packaging vector encoding a transgene of interest, (b) potassium chloride at a concentration of 0.2 g / L, (c) potassium phosphate monobasic at a concentration of 0.2 g / L, (d) sodium chloride at a concentration of 5.84 g / L, (e) sodium phosphate dibasic anhydrous at a concentration of 1.15 g / L, (f) sucrose at a concentration of 4% weight / volume (40 g / L), (g) poloxamer 188 at a concentration of 0.001% weight / volume (0.01 g / L), and (h) water, wherein the transgene of interest encodes an RNA of interest or a protein of interest, e.g., human TPP1.

[0151] In some embodiments, the pharmaceutical composition is a liquid composition. In some embodiments, the pharmaceutical composition is a frozen composition. In some embodiments, the pharmaceutical composition is a lyophilized composition from a liquid composition disclosed herein. In some embodiments, the pharmaceutical composition is a reconstituted lyophilized formulation.

[0152] In some embodiments, the pharmaceutical composition is a lyophilized composition having a residual moisture content of about 1% to about 7%. In some embodiments, the pharmaceutical composition is a lyophilized composition having a residual moisture content of about 2% to about 6%. In some embodiments, the pharmaceutical composition is a lyophilized composition having a residual moisture content of about 3% to about 4%. In some embodiments, the pharmaceutical composition is a lyophilized composition having a residual moisture content of about 5%.

[0153] In certain aspects, disclosed herein are methods for treating or preventing a disease in a subject, the methods comprising administering to the subject a pharmaceutical composition. In some embodiments, the pharmaceutical compositions provided herein are suitable for administration by one, two, or more routes of administration (e.g., suitable for suprachoroidal and subretinal administration).

[0154] The provided methods are suitable for use in producing pharmaceutical compositions comprising recombinant AAV encoding a transgene. In some embodiments, provided herein is an rAAV viral vector encoding tripeptidyl peptidase 1 (TPP1) protein. In some embodiments, provided herein is an rAAV9-based viral vector encoding TPP1. In some embodiments, provided herein is an rAAV9-based viral vector encoding CLN2.

[0155] In certain aspects, disclosed herein are methods of treating or preventing a disease in a subject, comprising administering to the subject a pharmaceutical composition by intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via a suprachoroidal drug delivery device such as a microinjector with a microneedle), subretinal injection via a transvitreal approach (a surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure via a subretinal drug delivery device comprising a catheter that can be inserted into the suprachoroidal space and threaded towards the posterior pole, where a small needle injects into the subretinal space), and / or a posterior juxtascleral depot procedure (e.g., via a juxtascleral drug delivery device comprising a cannula whose tip can be inserted and maintained in direct apposition against the scleral surface).

[0156] In certain embodiments, the pharmaceutical compositions provided herein are suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via a suprachoroidal drug delivery device such as a microinjector with a microneedle), subretinal injection via a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure via a subretinal drug delivery device comprising a catheter that can be inserted into the suprachoroidal space and threaded towards the posterior pole, where a small needle injects into the subretinal space), and / or a posterior juxtascleral depot procedure (e.g., via a juxtascleral drug delivery device comprising a cannula whose tip can be inserted and maintained in direct apposition against the scleral surface).

[0157] In certain embodiments, the pharmaceutical composition has a desired viscosity suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via a suprachoroidal drug delivery device such as a microinjector with a microneedle), subretinal injection via a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure via a subretinal drug delivery device comprising a catheter that can be inserted into the suprachoroidal space and threaded towards the posterior pole, where a small needle injects into the subretinal space), and / or a posterior juxtascleral depot procedure (e.g., via a juxtascleral drug delivery device comprising a cannula whose tip can be inserted and maintained in direct apposition against the scleral surface).

[0158] In certain embodiments, the pharmaceutical composition has a desired density suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via a suprachoroidal drug delivery device such as a microinjector with a microneedle), subretinal injection via a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space (e.g., a surgical procedure via a subretinal drug delivery device comprising a catheter that can be inserted into the suprachoroidal space and threaded towards the posterior pole, where a small needle injects into the subretinal space), and / or a posterior juxtascleral depot procedure (e.g., via a juxtascleral drug delivery device comprising a cannula whose tip can be inserted and maintained in direct apposition against the scleral surface).

[0159] In certain embodiments, the pharmaceutical composition has a desired osmolality suitable for intravenous administration, subcutaneous administration, intramuscular injection, suprachoroidal injection (e.g., via a suprachoroidal drug delivery device such as a microinjector with a microneedle), subretinal injection via a transvitreal approach (surgical procedure), subretinal administration via the suprachoroidal space (e.g., via a subretinal drug delivery device including a catheter that can be inserted into the suprachoroidal space and threaded toward the posterior pole, where a small needle injects into the subretinal space), and / or a posterior juxtascleral depot procedure (e.g., via a juxtascleral drug delivery device with a cannula that can be inserted and maintained in direct apposition to the scleral surface). In certain embodiments, the desired osmolality for subretinal administration is 160-430 mOsm / kg HO. In other specific embodiments, the desired osmolality for suprachoroidal administration is less than 600 mOsm / kg HO.

[0160] In certain embodiments, the pharmaceutical composition has an osmolality of about 100-500 mOsm / kg HO. In certain embodiments, the pharmaceutical composition has an osmolality of about 130-470 mOsm / kg HO. In certain embodiments, the pharmaceutical composition has an osmolality of about 160-430 mOsm / kg HO. In certain embodiments, the pharmaceutical composition has an osmolality of about 200-400 mOsm / kg HO. In certain embodiments, the pharmaceutical composition has an osmolality of about 240-340 mOsm / kg HO. In certain embodiments, the pharmaceutical composition has an osmolality of about 280-300 mOsm / kg HO. In certain embodiments, the pharmaceutical composition has an osmolality of about 295-395 mOsm / kg HO. In certain embodiments, the pharmaceutical composition has an osmolality of less than 600 mOsm / kg HO. In certain embodiments, the pharmaceutical composition has an osmolality range of 200 mOsm / L to 660 mOsm / L. In certain embodiments, the pharmaceutical composition has an osmolality of about 200 mOsm / L. In certain embodiments, the pharmaceutical composition has an osmolality of about 250 mOsm / L. In certain embodiments, the pharmaceutical composition has an osmolality of about 300 mOsm / L. In certain embodiments, the pharmaceutical composition has an osmolality of about 350 mOsm / L. In certain embodiments, the pharmaceutical composition has an osmolality of about 400 mOsm / L. In certain embodiments, the pharmaceutical composition has an osmolality of about 450 mOsm / L. In certain embodiments, the pharmaceutical composition has an osmolality of about 500 mOsm / L. In certain embodiments, the pharmaceutical composition has an osmolality of about 550 mOsm / L. In certain embodiments, the pharmaceutical composition has an osmolality of about 600 mOsm / L. In certain embodiments, the pharmaceutical composition has an osmolality of about 650 mOsm / L. In certain embodiments, the pharmaceutical composition has an osmolality of about 660 mOsm / L.

[0161] The recombinant vector used to deliver the transgene should have tropism for human retinal or photoreceptor cells. Such vectors can include non-replicating recombinant adeno-associated viral vectors ("rAAV"), although other viral vectors may also be used, including, but not limited to, lentiviral vectors, vaccinia viral vectors, or non-viral expression vectors referred to as "naked DNA" constructs.

[0162] In certain embodiments, the gene therapy construct is supplied as a freeze-sterilized single-use solution of the AAV vector active ingredients in a formulation buffer. In certain embodiments, a pharmaceutical composition suitable for subretinal administration comprises a suspension of the recombinant vector in a formulation buffer comprising a physiologically compatible aqueous buffer, a surfactant, and optional excipients. In certain embodiments, the construct is formulated in Dulbecco's phosphate-buffered saline and 0.001% poloxamer 188 (pH=7.4).

[0163] 4.3 Gene therapy

[0009] In one aspect, provided herein are methods for treating and / or preventing ocular symptoms of CLN2 Batten disease in a subject, the method comprising administering to the eye of a subject in need thereof a recombinant adeno-associated virus (rAAV) comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising (a) an AAV 5' inverted terminal repeat (ITR) sequence, (b) a promoter, (c) a CLN2 coding sequence encoding human TPP1, and (d) an AAV 3' ITR. In some embodiments, the rAAV is administered subretinally. In some embodiments, the rAAV is administered suprachoroidally.

[0164] In one aspect, provided herein are methods for treating and / or preventing ocular symptoms of CLN2 Batten disease in a subject, the method comprising subretinal administration to a subject in need thereof of a recombinant adeno-associated virus (rAAV) comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising (a) an AAV 5' inverted terminal repeat (ITR) sequence, (b) a promoter, (c) a CLN2 coding sequence encoding human TPP1, and (d) an AAV 3' ITR; the method does not comprise performing a vitrectomy on the eye of the human patient. In certain embodiments, the administering step comprises administering a recombinant viral vector therapeutic product to the subretinal space of the eye of the human subject via the suprachoroidal space of the eye of the human subject. In certain embodiments, the administering step is by use of a subretinal drug delivery device comprising a catheter insertable into the suprachoroidal space and threadable through the suprachoroidal space toward the posterior pole, where a fine needle is injected into the subretinal space. In certain embodiments, the administering step comprises inserting and passing a catheter of the subretinal drug delivery device into and through the suprachoroidal space.

[0165] In another aspect, provided herein are methods of treating and / or preventing ocular symptoms of CLN2 Batten disease in a subject, the method comprising subretinal administration to a subject in need thereof of a recombinant adeno-associated virus (rAAV) comprising an AAV capsid and, packaged therein, a vector genome, the vector genome comprising (a) an AAV 5' inverted terminal repeat (ITR) sequence, (b) a promoter, (c) a CLN2 coding sequence encoding human TPP1, and (d) an AAV 3' ITR; the method also comprises performing a vitrectomy on the eye of the human patient. In certain embodiments, the vitrectomy is a partial vitrectomy.

[0166] In another aspect, provided herein are methods for treating and / or preventing ocular symptoms of CLN2 Batten disease in a subject, the method comprising suprachoroidally administering to a subject in need thereof a recombinant adeno-associated virus (rAAV) comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising (a) an AAV 5' inverted terminal repeat (ITR) sequence, (b) a promoter, (c) a CLN2 coding sequence encoding human TPP1, and (d) an AAV 3' ITR. In certain embodiments, the administering step is by injecting the recombinant viral vector into the suprachoroidal space using a suprachoroidal drug delivery device. In certain embodiments, the suprachoroidal drug delivery device is a microinjector.

[0167] In another aspect, provided herein are methods for treating and / or preventing ocular symptoms of CLN2 Batten disease in a subject, the method comprising administering to the outer surface of the sclera of a subject in need thereof a recombinant adeno-associated virus (rAAV) comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising: (a) an AAV 5' inverted terminal repeat (ITR) sequence; (b) a promoter; (c) a CLN2 coding sequence encoding human TPP1; and (d) an AAV 3' ITR. In certain embodiments, the administering step is by use of a juxtascleral drug delivery device comprising a cannula, the tip of which can be inserted into and directly apposed to the scleral surface. In certain embodiments, the administering step comprises inserting the tip of the cannula into and directly apposing the scleral surface.

[0168] In another aspect, provided herein are methods for treating and / or preventing ocular symptoms of CLN2 Batten disease in a subject, the method comprising intravitreally administering to a subject in need thereof a recombinant adeno-associated virus (rAAV) comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising (a) an AAV 5' inverted terminal repeat (ITR) sequence, (b) a promoter, (c) a CLN2 coding sequence encoding human TPP1, and (d) an AAV 3' ITR. In certain embodiments, the administering step is by injecting the recombinant viral vector into the vitreous cavity using an intravitreal drug delivery device. In certain embodiments, the intravitreal drug delivery device is a microinjector.

[0169] In another aspect, provided herein are methods for treating and / or preventing ocular symptoms of CLN2 Batten disease in a subject, the method comprising administering to the vitreous cavity of the eye of a subject in need thereof a recombinant adeno-associated virus (rAAV) comprising an AAV capsid and a vector genome packaged therein, the vector genome comprising (a) an AAV 5' inverted terminal repeat (ITR) sequence, (b) a promoter, (c) a CLN2 coding sequence encoding human TPP1, and (d) an AAV 3' ITR. In certain embodiments, the administering step is by injecting the recombinant viral vector into the vitreous cavity using an intravitreal drug delivery device. In certain embodiments, the intravitreal drug delivery device is a microinjector.

[0170]

[0009] In one aspect, provided herein is a method of subretinal administration accompanied by vitrectomy for treating and / or preventing ocular symptoms of CLN2 Batten disease, comprising administering an rAAV to the subretinal space of the eye of a human subject in need of treatment, the method comprising performing vitrectomy on the eye of the human patient, wherein the rAAV comprises an AAV capsid and a vector genome packaged therein, the vector genome comprising (a) an AAV 5' inverted terminal repeat (ITR) sequence, (b) a promoter, (c) a CLN2 coding sequence encoding human TPP1, and (d) an AAV 3' ITR. In certain embodiments, the vitrectomy is a partial vitrectomy.

[0171] In one aspect, provided herein is a method of subretinal administration for treating and / or preventing ocular symptoms of CLN2 Batten disease, comprising administering an rAAV to the subretinal space surrounding the optic nerve head, fovea, and macula located in the posterior segment of the eye of a human subject in need of treatment, the method not comprising performing a vitrectomy on the eye of the human patient, wherein the rAAV comprises an AAV capsid and a vector genome packaged therein, the vector genome comprising (a) an AAV 5' inverted terminal repeat (ITR) sequence, (b) a promoter, (c) a CLN2 coding sequence encoding human TPP1, and (d) an AAV 3' ITR. In certain embodiments, the injection step is by intravitreal injection. In certain embodiments, the intravitreal administration method results in uniform expression of the therapeutic product throughout the eye (e.g., expression levels at the injection site vary by less than 5%, 10%, 20%, 30%, 40%, or 50% compared to expression levels in other regions of the eye). In certain embodiments, the transvitreal injection involves inserting a sharp needle into the sclera via the superior or inferior side of the eye, passing the sharp needle completely through the vitreous body, and injecting the recombinant viral vector into the subretinal space on the opposite side. In certain embodiments, the needle is inserted at the 2 o'clock or 10 o'clock position. In certain embodiments, the transvitreal injection involves inserting a trocar into the sclera, inserting a cannula into the trocar, and injecting the recombinant viral vector through the vitreous body into the subretinal space on the opposite side.

[0172] In certain embodiments of the methods described herein, the administering step delivers a therapeutically effective amount of rAAV to the retina of the human subject.

[0173] In certain embodiments of the methods described herein, a therapeutically effective amount of a protein encoded by the rAAV genome (e.g., TPP1) is produced by human retinal cells of the human subject.

[0174] In certain embodiments of the methods described herein, a therapeutically effective amount of a protein encoded by the rAAV genome (e.g., TPP1) is produced by human photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, and / or retinal pigment epithelial cells in the external limiting membrane of the human subject.

[0175] In certain embodiments of the methods described herein, the human photoreceptor cells are cone cells and / or rod cells.

[0176] In certain embodiments of the methods described herein, the retinal ganglion cells are midget cells, parasol cells, bistratified cells, giant retinal ganglion cells, photosensitive ganglion cells, and / or Müller glia.

[0177] In certain embodiments of the methods described herein, the recombinant viral vector is an rAAV vector (e.g., an rAAV8, rAAV2, rAAV9, or rAAV5 vector).

[0178] In certain embodiments of the methods described herein, the recombinant viral vector is an rAAV9 vector.

[0179] In certain embodiments of the methods described herein, delivery to the eye includes delivery to the retina, choroid, and / or vitreous humor of the eye. Methods are described for administering a therapeutically effective amount of a recombinant vector (i.e., a recombinant viral vector or a DNA expression construct) to a human subject having an ocular condition. In some embodiments, the recombinant vector is Construct I, which comprises the following components: an AAV capsid and a vector genome packaged therein, the vector genome comprising (a) an AAV 5' inverted terminal repeat (ITR) sequence, (b) a promoter, (c) a CLN2 coding sequence encoding human TPP1, and (d) an AAV 3' ITR. In some embodiments, the 5' and / or 3' ITRs are derived from AAV2. In some embodiments, the promoter is a chicken beta-actin promoter. In some embodiments, the methods provided herein are for treating ocular symptoms of Batten disease, including, but not limited to, vision loss, retinal atrophy, and retinal degeneration.

[0180] In particular, methods are described for administering a therapeutically effective amount of a recombinant vector (i.e., a recombinant viral vector or a DNA expression construct) to a human subject via one of the following approaches: (1) subretinal administration without vitrectomy (e.g., administration via the suprachoroidal space or into the subretinal space via periclinal injection), (2) suprachoroidal administration, (3) administration into the space outside the sclera (i.e., juxtascleral administration), (4) subretinal administration with vitrectomy, (5) intravitreal administration, and (6) subconjunctival administration.

[0181] In certain embodiments, delivery to the subretinal or suprachoroidal space can be achieved using the methods and / or devices described and disclosed in WO 2016 / 042162, WO 2017 / 046358, WO 2017 / 158365, and WO 2017 / 158366, each of which is incorporated herein by reference in its entirety.

[0182] 4.3.1 Target patient population In certain embodiments of the methods described herein, the methods provided herein are for administration to a patient with ocular symptoms associated with Batten disease, including, but not limited to, progressive vision loss, retinal atrophy and / or retinal degeneration.

[0183] In certain embodiments of the methods described herein, the human subject has a BCVA that is ≦20 / 20 and ≧20 / 400. In another specific embodiment, the human subject has a BCVA that is ≦20 / 63 and ≧20 / 400.

[0184] In some embodiments, the human subject has a biallelic CLN2 mutation.

[0185] In some embodiments, the human subject has reduced leukocyte TPP1 activity.

[0186] In some embodiments, the human subject has clinical signs or symptoms consistent with CLN2 disease (e.g., developmental delay, developmental decline, seizures, vision loss, or other symptoms) and / or the subject has an older sibling with a confirmed CLN2 diagnosis.

[0187] In some embodiments, the human subject has undergone or is undergoing intraventricular Brineura enzyme replacement therapy.

[0188] In some embodiments, the human subject has a CRT of about or less than about 210 μm in one or both eyes, hi some embodiments, the human subject has a CRT of about or greater than about 140 μm in one or both eyes.

[0189] In some embodiments, the human subject, when CLN2 disease is diagnosed in an asymptomatic patient due to a family history of CLN2 disease in siblings, the onset of vision loss in the affected sibling occurs before or around the age of 84 months.

[0190] In some embodiments, the human subject has a CRT of about 140 μm or less than about 140 μm in one or both eyes, and a Weill Cornell Ophthalmology Severity Score greater than 5.

[0191] In some embodiments, the human subject does not have significant lens or corneal opacities, glaucoma, amblyopia, and / or gross retinal anatomical abnormalities.

[0192] In some embodiments, the human subject has no more than a 10 μm difference in screening CRT measurements between the right and left eyes.

[0193] In some embodiments, the human subject has no previous grade 3 or 4 hypersensitivity reactions, e.g., bronchospasm and hypotension requiring intravenous treatment, cardiac dysfunction, or anaphylaxis to intraventricular Brineura infusion.

[0194] In some embodiments, the human subject does not have refractive errors in the following ranges (myopia >=-2.00, hyperopia >=+4.00, astigmatism >=+1.50) or significant anisometropia.

[0195] In some embodiments, the human subject has not received any kind of prior intraocular injection, for example, off-label use of intravitreal Brineura.

[0196] In some embodiments, the human subject has not undergone ophthalmic surgery, for example, within six months prior to starting Construct I treatment.

[0197] In some embodiments, the human subject has not previously undergone a bone marrow transplant.

[0198] In some embodiments, the human subject has not used the following medications within 30 days prior to starting Construct I treatment: gemfibrozil, mycophenolate, prednisone, or other steroids for the intended purpose of treating NCL (not including the indication for asthma), flupirtine.

[0199] In some embodiments, the human subject has no contraindications to systemic immunosuppression.

[0200] In some embodiments, the human subject does not have a mutation in another CLN gene.

[0201] In some embodiments, the human subject does not have a contraindication to intraocular surgery (e.g., a severe coagulation disorder).

[0202] In certain embodiments, the subject treated according to the methods described herein is female.In certain embodiments, the subject treated according to the methods described herein is male.In certain embodiments, the subject treated according to the methods described herein is a child.In certain embodiments, the subject treated according to the methods described herein is 1 month old, 2 months old, 3 months old, 4 months old, 5 months old, 6 months old, 7 months old, 8 months old, 9 months old, 10 months old, 11 months old, 1 year old, 1.5 years old, 2 years old, 2.5 years old, 3 years old, 3.5 years old, 4 years old, 4.5 years old or 5 years old. In certain embodiments, the subject treated according to the methods described herein is 1.5 months old, 2 months old, 3 months old, 4 months old, 5 months old, 6 months old, 7 months old, 8 months old, 9 months old, 10 months old, 11 months old, 1 year old, 1.5 years old, 2 years old, 2.5 years old, 3 years old, 3.5 years old, 4 years old, under 4.5 years old, or under 5 years old. In another specific embodiment, the subject treated in accordance with the methods described herein is 1 to 2 months old, 2 to 3 months old, 3 to 4 months old, 4 to 5 months old, 5 to 6 months old, 6 to 7 months old, 7 to 8 months old, 8 to 9 months old, 9 to 10 months old, 10 to 11 months old, 11 months old to 1 year old, 1 to 1.5 years old, 1.5 to 2 years old, 2 to 2.5 years old, 2.5 to 3 years old, 3 to 3.5 years old, 3.5 to 4 years old, 4 to 4.5 years old, or 4.5 to 5 years old. In another specific embodiment, the subject treated in accordance with the methods described herein is 6 months old to 5 years old. In another embodiment, the subject treated in accordance with the methods described herein is a human adult 18 years old or older. In some embodiments, the subject treated in accordance with the methods described herein is a human child under 18 years old. In some embodiments, the subject treated in accordance with the methods described herein is a human child under 84 months old.

[0203] 4.3.2 Dosage and Mode of Administration In certain embodiments of the methods described herein, a therapeutically effective dose of the recombinant vector is administered (1) to the subretinal space without vitrectomy (e.g., via the suprachoroidal space or via perichoroidal injection), (2) to the suprachoroidal space, (3) to the space outside the sclera (i.e., juxtascleral administration), (4) to the subretinal space via vitrectomy, or (5) to the vitreous space in a volume ranging from 50 to 100 μl or 100 to 500 μl, preferably 100 to 300 μl, and most preferably 250 μl, depending on the method of administration. In certain embodiments, a therapeutically effective dose of the recombinant vector is administered suprachoroidally in a volume of 100 μl or less, e.g., 50 to 100 μl. In certain embodiments, a therapeutically effective dose of the recombinant vector is administered to the outer surface of the sclera (e.g., via a posterior juxtascleral depot procedure) in a volume of 500 μl or less, e.g., 10-20 μl, 20-50 μl, 50-100 μl, 100-200 μl, 200-300 μl, 300-400 μl, or 400-500 μl. In certain embodiments, a therapeutically effective dose of the recombinant vector is administered to the subretinal space via a periapical injection in a volume of 50-100 μl or 100-500 μl, preferably 100-300 μl, and most preferably 200 μl.

[0204] In certain embodiments, a therapeutically effective dose of the recombinant vector is administered subretinally in a volume of 200 μL.

[0205] In certain embodiments, described herein is a microvolume injector delivery system manufactured by Altaviz (see, e.g., International Patent Application Publication No. 2013 / 177215, U.S. Patent Application Publication No. 2019 / 0175825, and U.S. Patent Application Publication No. 2019 / 0167906), which can be used for any of the routes of administration described herein for ocular administration. The microvolume injector delivery system can include a gas drive module that provides high-force delivery and improved precision, as described in U.S. Patent Application Publication Nos. 2019 / 0175825 and 2019 / 0167906. Additionally, the microvolume injector delivery system can include a hydraulic drive to provide a constant dose rate and a low-force actuation lever to control the gas drive module and, therefore, fluid delivery.

[0206] In certain embodiments, the microvolume injector delivery system can be a microvolume injector with dose guidance, for example, a suprachoroidal needle (e.g., a Clearside® needle), a subretinal needle, an intravitreal needle, a juxtascleral needle, a subconjunctival needle, and / or an intraretinal needle. Advantages of using a microvolume injector include: (a) more controlled delivery (e.g., due to precise injection flow rate control and dose guidance); (b) single-surgeon, single-hand, single-finger operation; (c) pneumatic actuation of 10 μL incremental doses; (d) freedom from vitrectomy; (e) 400 μL syringe dose; (f) digitally guided delivery; (g) digitally recorded delivery; and (h) a less restrictive tip (e.g., a MedOne 38 g needle and a Dorc 41 g needle can be used for subretinal delivery, while a Clearside® needle and a Visionisti OY adapter can be used for subretinal delivery).

[0207] In certain embodiments of the methods described herein, the recombinant vector is administered suprachoroidally (e.g., by suprachoroidal injection). In certain embodiments, suprachoroidal administration (e.g., injection into the suprachoroidal space) is performed using a suprachoroidal drug delivery device. Suprachoroidal drug delivery devices are often used in suprachoroidal administration procedures, which involve administering a drug to the suprachoroidal space of the eye (see, for example, Hariprasad, 2016, Retinal Physician 13:20-23; Goldstein, 2014, Retina Today 9(5):82-87; Baldassarre et al., 2017 (each of which is incorporated herein by reference in its entirety)). Suprachoroidal drug delivery devices that can be used to deposit recombinant vectors into the suprachoroidal space in accordance with the invention described herein include, but are not limited to, the suprachoroidal drug delivery device manufactured by Clearside® Biomedical, Inc. (see, e.g., Hariprasad, 2016, Retinal Physician 13:20-23) and the MedOne suprachoroidal catheter. In another embodiment, a suprachoroidal drug delivery device that can be used in accordance with the methods described herein includes a microvolume injector delivery system manufactured by Altaviz, which can be used for any of the routes of administration described herein for ocular administration (see, e.g., International Patent Application Publication No. 2013 / 177215, U.S. Patent Application Publication No. 2019 / 0175825, and U.S. Patent Application Publication No. 2019 / 0167906). The microvolume injector delivery system may include a gas drive module that provides high-force delivery and improved precision, as described in U.S. Patent Application Publication Nos. 2019 / 0175825 and 2019 / 0167906. Additionally, the microvolume injector delivery system may include a hydraulic drive to provide a constant dose rate and a low-force actuation lever to control the gas drive module and thus fluid delivery. Currently available technologies exist for suprachoroidal space (SCS) delivery. Preclinically, SC injections have been achieved with scleral flap techniques, catheters, and standard hypodermic needles, as well as microneedles.A hollow, 750-um-long microneedle (Clearside Biomedical, Inc.) can be inserted into the submucosal cavity and has shown promise in clinical trials. Microneedles designed using force-measuring technology can be utilized for SC injections, as described by Chitnis et al. (Chitnis, GD, et al. A resistance-sensing mechanical injector for the precise delivery of liquids to target tissue. Nat Biomed Eng 3, 621-631 (2019). https: / / doi.org / 10.1038 / s41551-019-0350-2). Oxular Limited is developing a delivery system (Oxulumis) that advances an illuminated cannula into the suprachoroidal space. The Orbit device (Gyroscope) is a specially designed system that enables cannulation of the suprachoroidal space with a flexible cannula. A microneedle within the cannula can be advanced into the subretinal space, enabling targeted dose delivery. Intraocular access to the SCS can also be achieved using microstents, which function as minimally invasive glaucoma surgery (MIGS) devices. Examples include the CyPass® microstent (Alcon, Fort Worth, Texas, US) and the iStent® (Glaukos), which are surgically implanted to provide a conduit from the anterior chamber to the SCS to drain aqueous humor without forming a filtering bleb. Other devices intended for suprachoroidal delivery include those described in British Patent Publication No. GB ​​2531910A and U.S. Patent No. 10,912,883 B2.

[0208] The microvolume injector is a microvolume injector with dose guidance and can be used, for example, with a suprachoroidal needle (e.g., a Clearside® needle) or a subretinal needle. Advantages of using a microvolume injector include: (a) more controlled delivery (e.g., due to precise injection flow rate control and dose guidance); (b) single-surgeon, single-hand, single-finger operation; (c) pneumatic actuation of 10 μL incremental doses; (d) freedom from vitrectomy; (e) 400 μL syringe dose; (f) digitally guided delivery; (g) digitally recorded delivery; and (h) a less restrictive tip (e.g., the MedOne 38 g needle and Dorc 41 g needle can be used for subretinal delivery, while the Clearside® needle and Visionisti OY adapter can be used for suprachoroidal delivery). In another embodiment, a suprachoroidal drug delivery device that can be used in accordance with the methods described herein is a tool manufactured by Visionisti OY, comprising a standard-length hypodermic needle with an adapter (preferably also a needle guide), which converts the standard-length hypodermic needle into a suprachoroidal needle by controlling the length of the needle tip exposed from the adapter (see, e.g., U.S. Design Patent No. D878,575; and International Patent Application Publication No. 2016 / 083669). In a specific embodiment, the suprachoroidal drug delivery device is a syringe with a 1-millimeter 30-gauge needle. During injection using this device, the needle penetrates the base of the sclera, and drug-containing fluid enters the suprachoroidal space, leading to expansion of the suprachoroidal space. This results in tactile and visual feedback during injection. After injection, the fluid flows backward and is primarily absorbed by the choroid and retina. This allows the therapeutic product to be produced from all retinal cell layers and choroidal cells. This type of device and procedure allows for a quick, easy in-office procedure with a low risk of complications. A maximum volume of 100 μl can be injected into the suprachoroidal space.

[0209] In certain embodiments of the methods described herein, the recombinant vector is administered subretinal via vitrectomy. Subretinal administration via vitrectomy is a surgical procedure performed by a trained retinal surgeon and involves vitrectomy in a subject under local anesthesia and subretinal injection of gene therapy into the retina (see, e.g., Campochiaro et al., 2017, Hum Gen Ther 28(1):99-111, incorporated herein by reference in its entirety).

[0210] In certain embodiments of the methods described herein, the recombinant vector is administered subretinally without vitrectomy.

[0211] In certain embodiments of the methods described herein, subretinal administration without vitrectomy is achieved via the suprachoroidal space through the use of a subretinal drug delivery device. In certain embodiments, the subretinal drug delivery device is a catheter inserted into and through the suprachoroidal space around the posterior segment of the eye during a surgical procedure to deliver drugs to the subretinal space. This procedure leaves the vitreous intact, reducing the risk of complications (such as gene therapy withdrawal and retinal detachment and macular holes), and without vitrectomy, the resulting bleb may spread more diffusely, allowing a greater surface area of ​​the retina to be transduced with a smaller volume. The risk of cataract induction after this procedure is minimized, which is desirable for younger patients. Furthermore, this procedure allows for safer subfoveal delivery of the bleb than standard transvitreal approaches, which is desirable for patients with inherited retinal diseases affecting central vision, where the target cells for transduction are in the macula. This procedure is also preferred for patients with neutralizing antibodies (NABs) against AAV present in the systemic circulation, which may affect other delivery routes (e.g., suprachoroidal and intravitreal). Additionally, this method has been shown to produce blebs with less release from the retinotomy site than standard transvitreal approaches. Subretinal drug delivery devices (see, e.g., Subretinal Delivery of Cells via the Suprachoroidal Space: Janssen Trial. In: Schwartz et al. (eds) Cellular Therapies for Retinal Disease, Springer, Cham, WO 2016 / 040635 A1) originally manufactured by Janssen Pharmaceuticals, Inc. and now by Orbit Biomedical Inc. can be used for this purpose.

[0212] In another specific embodiment, subretinal administration without vitrectomy is via a peripheral injection into the retina (i.e., around the optic disc, fovea, and macula, located in the posterior segment of the eye), which can be accomplished by intravitreal injection.

[0213] In one embodiment, a sharp needle is inserted into the sclera via the superior or inferior side of the eye (e.g., at the 2 o'clock or 10 o'clock position) and passed completely through the vitreous body to inject into the contralateral retina. In another embodiment, a trocar is inserted into the sclera to allow a subretinal cannula to be inserted into the eye. The cannula is inserted into the trocar and passed through the vitreous body to the desired injection area. In either embodiment, the recombinant vector is injected into the subretinal space, forming a bleb containing the recombinant vector on the inner surface of the contralateral eye. Successful injection is confirmed by the appearance of a dome-shaped retinal detachment / retinal bleb.

[0214] A self-illuminating lens may be used as a light source for transvitreal administration (see, e.g., Chalam et al., 2004, Ophthalmic Surgery and Lasers 35:76-77, incorporated herein by reference in its entirety). Alternatively, one or more trocars may be positioned for light (or injection) as needed. In yet another embodiment, a fiber optic chandelier may be utilized through the trocar to visualize the subretinal injection.

[0215] In certain embodiments, delivery to the subretinal or suprachoroidal space can be achieved using the methods and / or devices described and disclosed in International Publication Nos. WO2016 / 042162, WO2017 / 046358, WO2017 / 158365, and WO2017 / 158366, each of which is incorporated herein by reference in its entirety.

[0216] The recombinant vector can be administered using one, two, or more peri-injections. In this manner, one, two, or more blebs containing the recombinant vector can be created in the subretinal space around the optic disc, fovea, and macula. Surprisingly, although administration of the recombinant vector is limited to the peri-injection blebs, this approach allows for detection of therapeutic product expression throughout the retina.

[0217] In certain embodiments, intravitreal administration is performed using an intravitreal drug delivery device manufactured by Altaviz, including a microvolume injector delivery system (see, e.g., International Patent Application Publication No. 2013 / 177215, U.S. Patent Application Publication No. 2019 / 0175825, and U.S. Patent Application Publication No. 2019 / 0167906), which can be used for any of the routes of administration described herein for ocular administration. The microvolume injector delivery system can include a gas drive module that provides high-force delivery and improved precision, as described in U.S. Patent Application Publication Nos. 2019 / 0175825 and 2019 / 0167906. Additionally, the microvolume injector delivery system can include a hydraulic drive to provide a constant dose rate and a low-force actuation lever to control the gas drive module and, therefore, fluid delivery. The microvolume injector is a microvolume injector with dose guidance and can be used, for example, with an intravitreal needle. Advantages of using a microvolume injector include: (a) more controlled delivery (e.g., due to precise injection flow rate control and dose guidance), (b) single-surgeon, single-hand, single-finger operation; (c) pneumatic actuation of 10 μL incremental doses; (d) separation from the vitrectomy machine; (e) 400 μL syringe dose; (f) digitally guided delivery; (g) digitally recorded delivery; and (h) a less restrictive tip (e.g., MedOne 38 g needles and Dorc 41 g needles can be used for subretinal delivery, while Clearside® needles and Visionisti OY adapters can be used for subretinal delivery).

[0218] In certain embodiments, peri-injection results in uniform expression of the therapeutic product throughout the eye (e.g., the variation in expression levels at the injection site is less than 5%, 10%, 20%, 30%, 40%, or 50% compared to expression levels in other areas of the eye). Expression of the therapeutic product throughout the eye can be measured by any method known in the art for such purpose, for example, by whole-mount immunofluorescence staining of the eye or retina, or by immunofluorescence staining on frozen eye sections.

[0219] In the event that the recombinant vector is lost in the vitreous but not in the subretinal space after intravitreal injection, an optional vitrectomy can be performed to remove the recombinant vector injected into the vitreous. Subretinal injection with vitrectomy can then be performed to deliver 250 μl of the recombinant vector to the subretinal space. Alternatively, if some of the injected recombinant vector is deposited in the vitreous and vitrectomy is not performed to remove the recombinant vector from the vitreous, a catheter with an immobilized (e.g., covalently bound) anti-AAV antibody (e.g., anti-AAV9 antibody) can be inserted into the vitreous to capture and remove excess recombinant vector from the vitreous.

[0220] In certain embodiments, subretinal administration is performed using a subretinal drug delivery device manufactured by Altaviz, including a microvolume injector delivery system (see, e.g., International Patent Application Publication No. 2013 / 177215, U.S. Patent Application Publication No. 2019 / 0175825, and U.S. Patent Application Publication No. 2019 / 0167906), which can be used for any of the administration routes described herein for ocular administration. The microvolume injector delivery system can include a gas drive module that provides high-force delivery and improved precision, as described in U.S. Patent Application Publication Nos. 2019 / 0175825 and 2019 / 0167906. Additionally, the microvolume injector delivery system can include a hydraulic drive to provide a constant dose rate and a low-force actuation lever to control the gas drive module and thus fluid delivery. The microvolume injector is a microvolume injector with dose guidance and can be used, for example, with a subretinal needle. Advantages of using a microvolume injector include: (a) more controlled delivery (e.g., due to precise injection flow rate control and dose guidance), (b) one-surgeon, one-hand, one-finger operation; (c) pneumatic actuation of 10 μL incremental doses; (d) separation from the vitrectomy machine; (e) 400 μL syringe dose; (f) digitally guided delivery; (g) digitally recorded delivery; and (h) a less restrictive tip (e.g., MedOne 38 g needles and Dorc 41 g needles can be used for subretinal delivery, while Clearside® needles and Visionisti OY adapters can be used for suprachoroidal delivery).

[0221] In certain embodiments, the recombinant vector is administered to the outer surface of the sclera (e.g., by use of a juxtascleral drug delivery device including a cannula, the tip of which can be inserted into the scleral surface and placed directly in apposition to it). In certain embodiments, administration to the outer surface of the sclera is achieved using a posterior juxtascleral depot procedure, in which the drug is drawn into a blunt, curved cannula and then delivered to direct contact with the outer surface of the sclera without puncturing the eye. Specifically, a small incision is made in the exposed sclera, followed by insertion of the tip of the cannula. The curved portion of the cannula shaft is inserted, holding the tip of the cannula in direct apposition to the scleral surface. After full insertion of the cannula, the drug is slowly injected while maintaining gentle pressure along the top and sides of the cannula shaft with a sterile cotton swab. This delivery method avoids the risk of intraocular infection and retinal detachment, side effects often associated with injecting therapeutic agents directly into the eye.

[0222] In certain embodiments, juxtascleral administration is performed using a juxtascleral drug delivery device manufactured by Altaviz, including a microvolume injector delivery system (see, e.g., International Patent Application Publication No. 2013 / 177215, U.S. Patent Application Publication No. 2019 / 0175825, and U.S. Patent Application Publication No. 2019 / 0167906), which can be used for any of the administration routes described herein for ocular administration. The microvolume injector delivery system can include a gas drive module that provides high-force delivery and improved precision, as described in U.S. Patent Application Publication Nos. 2019 / 0175825 and 2019 / 0167906. Additionally, the microvolume injector delivery system can include a hydraulic drive to provide a constant dose rate and a low-force actuation lever to control the gas drive module and thus fluid delivery. The microvolume injector is a microvolume injector with dose guidance and can be used, for example, with a juxtascleral needle. Advantages of using a microvolume injector include: (a) more controlled delivery (e.g., due to having precise injection flow rate control and dose guidance); (b) one-surgeon, one-hand, one-finger operation; (c) pneumatic actuation of 10 μL incremental doses; (d) separation from the vitrectomy machine; (e) 400 μL syringe dose; (f) digitally guided delivery; (g) digitally recorded delivery; and (h) a less restrictive tip.

[0223] In certain embodiments, an infrared thermal camera can be used to detect changes in the thermal profile of the ocular surface following administration of a solution that is cooler than body temperature. This allows, for example, visualization of the solution's spread within the SCS, potentially determining whether administration was successful. This is because, in certain embodiments, the formulation containing the recombinant vector to be administered is first frozen, brought to room temperature (68-72°F), and thawed for a short period (e.g., at least 30 minutes) before administration. Thus, at the time of injection, the formulation is colder than the human eye (approximately 92°F), and in some cases even colder than room temperature. The drug is typically used within four hours of thawing, and the warmest solution will be at room temperature. In a preferred embodiment, the procedure is videotaped with infrared video.

[0224] Infrared thermal cameras can detect small changes in temperature. They capture infrared energy through a lens and convert that energy into an electronic signal. The infrared light is focused onto an infrared sensor array, which converts the energy into a thermal image. Infrared thermal cameras can be used with any of the administration routes described herein, including suprachoroidal, subretinal, subconjunctival, intravitreal, or administration using a slow infusion catheter into the suprachoroidal space. In a specific embodiment, the infrared thermal camera is a FLIR T530 infrared thermal camera. The FLIR T530 infrared thermal camera can capture small temperature differences with an accuracy of ±3.6°F. The camera has an infrared resolution of 76,800 pixels. The camera also utilizes a 24° lens, which captures a smaller field of view. The smaller field of view combined with the high infrared resolution contributes to a more detailed thermal profile of what the operator is imaging. However, other infrared cameras with different infrared resolutions and / or lenses with different degrees of magnification can be used, with different capabilities and accuracy for capturing small temperature changes.

[0225] In certain embodiments, the infrared thermal camera is a FLIR T420 infrared thermal camera. In certain embodiments, the infrared thermal camera is a FLIR T440 infrared thermal camera. In certain embodiments, the infrared thermal camera is a Fluke Ti400 infrared thermal camera. In certain embodiments, the infrared thermal camera is a FLIRE60 infrared thermal camera. In certain embodiments, the infrared thermal camera has an infrared resolution of 75,000 pixels or greater. In certain embodiments, the infrared thermal camera has a thermal sensitivity of 0.05°C or less at 30°C. In certain embodiments, the infrared thermal camera has a field of view (FOV) of 25° x 25° or less.

[0226] In certain embodiments, an iron filter is used in conjunction with an infrared thermal camera to detect changes in the thermal profile of the ocular surface. In a preferred embodiment, the use of an iron filter can produce a pseudocolor image, with the warmest or hottest areas colored white, intermediate temperatures red and yellow, and the coolest or coldest areas black. In certain embodiments, other types of filters can also be used to produce a pseudocolor image of the thermal profile.

[0227] The thermal profile for each administration method may be different. For example, in one embodiment, a successful suprachoroidal injection can be characterized by (a) a slow, wide radial spread of dark color, (b) an initial very dark color, and (c) a gradual change in the injectate to a lighter color, i.e., a temperature gradient indicated by the lighter color. In one embodiment, an unsuccessful suprachoroidal injection can be characterized by (a) a lack of spread of dark color and (b) a slight change in color localized to the injection site without distribution. In certain embodiments, a small localized temperature drop results from the cannula (cold temperature) contacting the ocular tissue (hot temperature). In one embodiment, a successful intravitreal injection can be characterized by (a) a lack of spread of dark color, (b) an initial change to a very dark color localized to the injection site, and (c) a gradual and uniform change to a darker color throughout the eye. In one embodiment, extraocular outflow can be characterized by (a) a rapid flow outside the outer surface of the eye, (b) an initial very dark color, and (c) a rapid change to a lighter color.

[0228] Because the therapeutic product is continuously produced (either under the control of a constitutive promoter or induced by hypoxia when a hypoxia-inducible promoter is used), maintaining a low concentration can be effective. Vitreous humor concentrations can be measured directly in patient samples of vitreous humor or fluid collected from the anterior chamber, or can be estimated and / or monitored by measuring the patient's serum concentration of the therapeutic product—the ratio of systemic exposure to the therapeutic product to vitreous exposure is approximately 1:90,000. (See, e.g., the aqueous humor and serum concentrations of ranibizumab reported in Xu L, et al., 2013, Invest. Opthal. Vis. Sci. 54:1621, pp. 1616-1624 and Table 5, p. 1623, incorporated herein by reference in their entireties.)

[0229] In certain embodiments, the dosage is measured by genome copies / ml or number of genome copies administered to the patient's eye (e.g., administered suprachoroidally, subretinally, intravitreally, juxtascleral, subconjunctivally, and / or intraretinal). In certain embodiments, the dosage is measured by 1×109 Genome copies / ml ~ 1 x 10 15 In a specific embodiment, 1 x 10 genome copies / ml is administered. 9 Genome copies / ml ~ 1 x 10 10 In another specific embodiment, 1 x 10 genome copies / ml is administered. 10 Genome copies / ml ~ 1 x 10 11 In another specific embodiment, 1 x 10 genome copies / ml is administered. 10 ~5×10 11 In another specific embodiment, 1 x 10 genome copies are administered. 11 Genome copies / ml ~ 1 x 10 12 In another specific embodiment, 1 x 10 genome copies / ml is administered. 12 Genome copies / ml ~ 1 x 10 13 In another specific embodiment, 1 x 10 genome copies / ml is administered. 13 Genome copies / ml ~ 1 x 10 14 In another specific embodiment, 1 x 10 genome copies / ml is administered. 14 Genome copies / ml ~ 1 x 10 15 In another specific embodiment, about 1 x 10 genome copies / ml is administered. 9 In another specific embodiment, about 1 x 10 genome copies / ml is administered. 10 In another specific embodiment, about 1 x 10 genome copies / ml is administered. 11 In another specific embodiment, about 1 x 10 genome copies / ml is administered. 12 In another specific embodiment, about 1 x 10 genome copies / ml is administered. 13 In another specific embodiment, about 1 x 10 genome copies / ml is administered. 14 In another specific embodiment, about 1 x 10 genome copies / ml is administered. 15 In one particular embodiment, 1 x 10 genome copies / ml is administered. 9 ~1×10 15 In certain embodiments, 1 x 10 genome copies are administered. 9 ~1×10 10 In another specific embodiment, 1 x 10 genome copies are administered. 10 ~1×1011 In another specific embodiment, 1 x 10 genome copies are administered. 10 ~5×10 11 In another specific embodiment, 1 x 10 genome copies are administered. 11 ~1×10 12 In another specific embodiment, 1 x 10 genome copies are administered. 12 ~1×10 13 In another specific embodiment, 1 x 10 genome copies are administered. 13 ~1×10 14 In another specific embodiment, 1 x 10 genome copies are administered. 13 ~1×10 14 In another specific embodiment, 1 x 10 genome copies are administered. 14 ~1×10 15 In another specific embodiment, about 1 x 10 genome copies are administered. 9 In another specific embodiment, about 1 x 10 genome copies are administered. 10 In another specific embodiment, about 1 x 10 genome copies are administered. 11 In another specific embodiment, about 1 x 10 genome copies are administered. 12 In another specific embodiment, about 1 x 10 genome copies are administered. 13 In another specific embodiment, about 1 x 10 genome copies are administered. 14 In another specific embodiment, about 1 x 10 genome copies are administered. 15 In certain embodiments, about 3.0 x 10 genome copies are administered. 13 In certain embodiments, up to about 3.0 x 10 genome copies / eye are administered. 13 Administer genome copies / eye.

[0230] In one particular embodiment, about 2.0×10 10 In certain embodiments, about 6.0 x 10 genome copies / eye are administered. 10 Administer genome copies / eye.

[0231] In one particular embodiment, about 1.0×10 10 ~2.0×10 10Genome copies / eye are administered by subretinal injection. In certain embodiments, about 2.0 x 10 10 ~3.0×10 10 Genome copies / eye are administered by subretinal injection. In certain embodiments, about 3.0 x 10 10 ~4.0×10 10 Genome copies / eye are administered by subretinal injection. In one particular embodiment, about 4.0 x 10 10 ~5.0×10 10 Genome copies / eye are administered by subretinal injection. In certain embodiments, about 5.0 x 10 10 ~6.0×10 10 Genome copies / eye are administered by subretinal injection. In one particular embodiment, about 6.0 x 10 10 ~7.0×10 10 Genome copies / eye are administered by subretinal injection. In one particular embodiment, about 7.0 x 10 10 ~8.0×10 10 Genome copies / eye are administered by subretinal injection. In one particular embodiment, about 8.0 x 10 10 ~9.0×10 10 Genome copies / eye are administered by subretinal injection. In one particular embodiment, about 9.0 x 10 10 ~1.0×10 11 Genome copies / eye are administered by subretinal injection. In certain embodiments, about 1.0 x 10 11 ~2.0×10 11 Genome copies / eye are administered by subretinal injection.

[0232] In certain specific embodiments, about 2.0×10 10 In certain embodiments, about 6.0 x 10 genome copies / eye are administered by subretinal injection. 10 Genome copies / eye are administered by subretinal injection. In some embodiments, the injection volume for subretinal injection is 200 μL.

[0233] In one particular embodiment, about 2×10 10 ~3×10 10Genome copies / eye are administered by suprachoroidal injection. In certain embodiments, about 3 x 10 10 ~4×10 10 Genome copies / eye are administered by suprachoroidal injection. In one particular embodiment, about 4 x 10 10 ~5×10 10 Genome copies / eye are administered by suprachoroidal injection. In one particular embodiment, about 5 x 10 10 ~6×10 10 Genome copies / eye are administered by suprachoroidal injection. In one particular embodiment, about 6 x 10 10 ~7×10 10 Genome copies / eye are administered by suprachoroidal injection. In one particular embodiment, about 7 x 10 10 ~8×10 10 Genome copies / eye are administered by suprachoroidal injection. In one particular embodiment, about 8 x 10 10 ~9×10 10 Genome copies / eye are administered by suprachoroidal injection. In one particular embodiment, about 9 x 10 10 ~1×10 11 In certain embodiments, about 1 x 10 genome copies / eye are administered by suprachoroidal injection. 11 ~2×10 11 Genome copies / eye are administered by suprachoroidal injection. In certain embodiments, about 2 x 10 11 ~3×10 11 Genome copies / eye are administered by suprachoroidal injection. In certain embodiments, about 3 x 10 11 ~4×10 11 Genome copies / eye are administered by suprachoroidal injection. In certain embodiments, about 4 x 10 11 ~5×10 11 Genome copies / eye are administered by suprachoroidal injection. In one particular embodiment, about 5 x 10 11 ~6×10 11 Genome copies / eye are administered by suprachoroidal injection. In one particular embodiment, about 6 x 10 11 ~7×10 11 Genome copies / eye are administered by suprachoroidal injection. In one particular embodiment, about 7 x 1011 ~8×10 11 Genome copies / eye are administered by suprachoroidal injection. In one particular embodiment, about 8 x 10 11 ~9×10 11 Genome copies / eye are administered by suprachoroidal injection. In one particular embodiment, about 9 x 10 11 ~1×10 12 Genome copies / eye are administered by suprachoroidal injection.

[0234] In one particular embodiment, about 2×10 10 Genome copies / eye are administered by suprachoroidal injection. In certain embodiments, about 3 x 10 10 Genome copies / eye are administered by suprachoroidal injection. In certain embodiments, about 4 x 10 10 Genome copies / eye are administered by suprachoroidal injection. In one particular embodiment, about 5 x 10 10 Genome copies / eye are administered by suprachoroidal injection. In one particular embodiment, about 6 x 10 10 Genome copies / eye are administered by suprachoroidal injection. In one particular embodiment, about 7 x 10 10 Genome copies / eye are administered by suprachoroidal injection. In one particular embodiment, about 8 x 10 10 Genome copies / eye are administered by suprachoroidal injection. In one particular embodiment, about 9 x 10 10 In certain embodiments, about 1 x 10 genome copies / eye are administered by suprachoroidal injection. 11 Genome copies / eye are administered by suprachoroidal injection. In certain embodiments, about 1.5 x 10 11 Genome copies / eye are administered by suprachoroidal injection. In certain embodiments, about 2 x 10 11 Genome copies / eye are administered by suprachoroidal injection. In certain embodiments, about 2.5 x 10 11 Genome copies / eye are administered by suprachoroidal injection. In certain embodiments, about 3 x 10 11 Genome copies / eye are administered by suprachoroidal injection.

[0235] In certain embodiments, a single suprachoroidal injection is administered. In certain embodiments, two suprachoroidal injections are administered. In certain embodiments, the injection volume for the suprachoroidal injection is 100 μl.

[0236] As used herein, and unless otherwise specified, the term "about" means within plus or minus 10% of a given value or range. In certain embodiments, the term "about" encompasses the exact number recited.

[0237] 4.3.3 Sampling and Efficacy Monitoring In certain embodiments, when a human subject has disease symptoms in both the CNS and the eye (e.g., when the human subject has Batten disease), the methods provided herein include administering a recombinant vector (i.e., a recombinant viral vector or a DNA expression construct) described herein to a human subject via both a central nervous system (CNS) delivery route and an ocular delivery route (e.g., an ocular delivery route described herein). In certain embodiments, the ocular delivery route is selected from one of the following: (1) subretinal administration without vitrectomy (e.g., administration via the suprachoroidal space or into the subretinal space via pericholecystectomy), (2) suprachoroidal administration, (3) administration into the space outside the sclera (i.e., juxtascleral administration), (4) subretinal administration with vitrectomy, (5) intravitreal administration, and (6) intravitreal administration. In certain embodiments, the CNS delivery route is selected from one of the following: intracerebroventricular (ICV) delivery, intracisternal (IC) delivery, or intrathecal lumbar (IT-L) delivery.

[0238] The effect of the methods provided herein on visual impairment can be measured by BCVA (best corrected visual acuity), intraocular pressure, slit lamp biomicroscopy, and / or indirect ophthalmoscopy.

[0239] In certain embodiments, the effect of the methods provided herein on visual impairment can be measured by whether the eye of a human patient treated by the methods described herein achieves a BCVA of greater than 43 letters after treatment (e.g., 46-50 weeks or 98-102 weeks after treatment). A BCVA of 43 letters corresponds to a 20 / 160 approximate Snellen equivalent visual acuity. In certain embodiments, the eye of a human patient treated by the methods described herein achieves a BCVA of greater than 43 letters after treatment (e.g., 46-50 weeks or 98-102 weeks after treatment).

[0240] In certain embodiments, the effect of the methods provided herein on visual impairment can be measured by whether the eye of a human patient treated by the methods described herein achieves a BCVA of greater than 84 letters after treatment (e.g., 46-50 weeks or 98-102 weeks after treatment). A BCVA of 84 letters corresponds to a 20 / 20 approximate Snellen equivalent visual acuity. In certain embodiments, the eye of a human patient treated by the methods described herein achieves a BCVA of greater than 84 letters after treatment (e.g., 46-50 weeks or 98-102 weeks after treatment).

[0241] The effect of the methods provided herein on physical changes to the eye / retina can be measured by SD-OCT (SD-Optical Coherence Tomography).

[0242] Efficacy may be monitored as measured by electroretinography (ERG).

[0243] The efficacy of the methods provided herein can be monitored by measuring signs of vision loss, infection, inflammation, and other safety events, such as retinal detachment.

[0244] Retinal thickness can be monitored to determine the effectiveness of the methods provided herein. Without being bound by any particular theory, retinal thickness can be used as a clinical readout, and the greater the reduction in retinal thickness or the longer the time until retinal thickening, the more effective the treatment. Retinal function can be determined, for example, by ERG. ERG is a non-invasive electrophysiological test of retinal function approved by the FDA for use in humans, which examines the light-sensitive cells (rods and cones) of the eye and their connected ganglion cells, particularly their response to flash stimuli. Retinal thickness can be determined, for example, by SD-OCT. SD-OCT is a three-dimensional imaging technique that uses low-coherence interferometry to determine the echo time delay and amplitude of backscattered light reflected from an object of interest. OCT can be used to scan layers of a tissue sample (e.g., the retina) with an axial resolution of 3 to 15 μm, and SD-OCT improves axial resolution and scanning speed over previous versions of the technique (Schuman, 2008, Trans. Am. Opthamol. Soc. 106:426-458).

[0245] The efficacy of the methods provided herein may also be measured by the change from baseline in the National Eye Institute Visual Function Questionnaire, Rasch score version (NEI-VFQ-28-R) (composite score, activity limitation domain score, and social-mental functioning domain score). The efficacy of the methods provided herein may also be measured by the change from baseline in the National Eye Institute Visual Function Questionnaire, 25-item version (NEI-VFQ-25) (composite score and mental health subscale score). The efficacy of the methods provided herein may also be measured by the change from baseline in the Macular Therapy Satisfaction Questionnaire (MacTSQ) (composite score; safety, efficacy, and discomfort domain scores; and informational and convenience domain scores).

[0246] In certain embodiments, the effectiveness of the methods described herein is reflected by an improvement in vision at about 4 weeks, 12 weeks, 6 months, 12 months, 24 months, 36 months, or other desired time points. In certain embodiments, the improvement in vision is characterized by an increase in BCVA, for example, an increase of 1 letter, 2 letters, 3 letters, 4 letters, 5 letters, 6 letters, 7 letters, 8 letters, 9 letters, 10 letters, 11 letters, or 12 letters or more. In certain embodiments, the improvement in vision is characterized by a 5%, 10%, 15%, 20%, 30%, 40%, 50% or more increase in visual acuity from baseline.

[0247] In certain embodiments, the effectiveness of the methods described herein is reflected by a reduction in central retinal thickness (CRT) or outer nuclear layer thickness (OLN) at about 4 weeks, 12 weeks, 6 months, 12 months, 24 months, 36 months, or other desired time point, e.g., a 5%, 10%, 15%, 20%, 30%, 40%, 50% or more reduction in central retinal thickness from baseline.

[0248] In certain embodiments, there is no inflammation in the eye after treatment, or there is little inflammation in the eye after treatment (e.g., inflammation levels increase by 10%, 5%, 2%, 1% or less from baseline).

[0249] The effect of the methods provided herein on visual impairment can be measured by optokinetic nystagmus (OKN). Without being bound by theory, this vision screening utilizes the principle of the OKN involuntary reflex to objectively assess whether a patient's eyes can track a moving target. Using the OKN, no verbal communication is required between the examiner and the patient. Therefore, the OKN can be used to measure the visual acuity of preverbal and / or non-verbal patients. In certain embodiments, the OKN is used to measure the visual acuity of patients who are 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, or 5 years old. In certain embodiments, an iPad is used to measure visual acuity through detection of the OKN reflex while the patient is viewing movements on the iPad. Without being bound by theory, this vision screening utilizes the principle of the OKN involuntary reflex to objectively assess whether a patient's eyes can track a moving target. By using the OKN, no verbal communication between the examiner and the patient is required. Therefore, the OKN can be used to measure the visual acuity of pre-verbal and / or non-verbal patients. In certain embodiments, the OKN is used to measure the visual acuity of patients who are under 1.5 months, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years, 4.5 years, or 5 years old. In another specific embodiment, the OKN is used to measure visual acuity in patients aged 1-2 months, 2-3 months, 3-4 months, 4-5 months, 5-6 months, 6-7 months, 7-8 months, 8-9 months, 9-10 months, 10-11 months, 11 months to 1 year, 1-1.5 years, 1.5-2 years, 2-2.5 years, 2.5-3 years, 3-3.5 years, 3.5-4 years, 4-4.5 years, or 4.5-5 years. In another specific embodiment, the OKN is used to measure visual acuity in patients aged 6 months to 5 years. In one specific embodiment, an iPad is used to measure visual acuity through detection of the OKN reflex while the patient watches movements on the iPad.

[0250] In certain embodiments, visual acuity is assessed in patients with Batten-CLN2-associated visual loss by measuring OKN before treating the patient with an AAV encoding tripeptidyl-peptidase 1 (TPP1), preferably AAV9. Specifically, the patient with Batten-CLN2-associated visual loss is of the age and / or within the age range described above. In certain embodiments, visual acuity is assessed in patients up to 5 years old with Batten-CLN2-associated visual loss by measuring OKN after treating the patient with an AAV encoding tripeptidyl-peptidase 1, preferably AAV9. In certain embodiments, visual acuity assessment based on OKN determines that visual acuity does not decrease after treatment with AAV gene therapy. In certain embodiments, the OKN-based visual acuity assessment determines that visual acuity improves by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% in the patient after treatment with AAV gene therapy. In certain embodiments, the OKN-based visual acuity assessment determines that visual acuity does not further deteriorate in the patient after treatment with AAV gene therapy. In certain embodiments, visual acuity is assessed in patients with Batten-CLN2-associated visual loss by measuring OKN before treating the patient with an AAV encoding TPP1, preferably AAV9. Specifically, the patient with Batten-CLN2-associated visual loss is of the age and / or within the age range described above. In certain embodiments, visual acuity is assessed in patients up to 5 years old with Batten-CLN2-associated visual loss by measuring OKN after treating the patient with an AAV encoding tripeptidyl-peptidase 1, preferably AAV9. In certain embodiments, visual acuity assessment based on OKN determines that visual acuity does not decrease after treatment with AAV gene therapy. In certain embodiments, the OKN-based visual acuity assessment determines that visual acuity improves by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or at least 100% in the patient following treatment with AAV gene therapy. In certain embodiments, the OKN-based visual acuity assessment determines that visual acuity does not further deteriorate in the patient following treatment with AAV gene therapy. If the human patient is a child, visual function can be assessed using an optokinetic nystagmus (OKN)-based approach or a modified OKN-based approach.

[0251] The efficacy of the methods provided herein can also be measured by a change in pupillary light reflex over time, as measured by pupillometry, for example, a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more change in pupillary light reflex from baseline at about 1-2, 2-4, 4-6, 8-10, 10-12, 12-14, 12-16, 16-18, 18-20, 20-22, 22-24 months, or at about 1, 3, 6, 9, 12, 15, 18, 21, or 24 months from baseline.

[0252] The efficacy of the methods provided herein can also be measured by a change in macular thickness and / or volume over time, e.g., a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more change in macular thickness and / or volume from baseline at about 1-2, 2-4, 4-6, 8-10, 10-12, 12-14, 12-16, 16-18, 18-20, 20-22, 22-24 months, or at about 1, 3, 6, 9, 12, 15, 18, 21, or 24 months from baseline, as measured by SD-OCT.

[0253] The efficacy of the methods provided herein can also be measured by a change in the time to an accelerated decline stage of retinal deterioration (e.g., reaching a 210 μm CRT), e.g., a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more change in the time to an accelerated decline stage of retinal deterioration, or to a 30 μm loss in CRT or to a CRT of less than about 110 μm (e.g., about 1-2, 2-4, 4-6, 8-10, 10-12, 12-14, 12-16, 16-18, 18-20, 20-22, 22-24 months, or about 1, 3, 6, 9, 12, 15, 18, 21, or 24 months) in the time to an accelerated decline stage of retinal deterioration, or to a 30 μm loss in CRT or to a CRT of less than about 110 μm.

[0254] The effects of the methods provided herein can also be evaluated by changes in SD-OCT anatomical markers, e.g., parafoveal ellipsoid zone, ELM, RNFL, total retinal pressure, inner nuclear layer, outer nuclear layer (ONL), photoreceptor (PR) + retinal pigment epithelium (RPE), outer segment + RPE (OS+RPE), and / or ellipsoid zone (EZ) over time, e.g., at about 1-2, 2-4, 4-6, 8-10, 10-12, 12-14, 12-16, 16-18, 18-20, 20-22, 22-24 months from baseline, or at about 1-2, 2-4, 4-6, 8-10, 10-12, 12-14, 12-16, 16-18, 18-20, 20-22, 22-24 months ...2-4, 2-4, 2-4, 2-4, 2-4, 2-4, 2-4, 2-4, 2-4, 2-4, 2-4, 2-4, 2-4, 2-4, 2-4, 2-4, 2-4, 2 It may also be measured by a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more change from baseline in SD-OCT anatomical markers, such as the parafoveal ellipsoid zone, ELM, RNFL, total retinal pressure, inner nuclear layer, outer nuclear layer (ONL), photoreceptor (PR) + retinal pigment epithelium (RPE), outer segment + RPE (OS+RPE), and / or ellipsoid zone (EZ) at 1, 3, 6, 9, 12, 15, 18, 21, or 24 months.

[0255] The efficacy of the methods provided herein can also be measured by a change over time in fundus photographic appearance as measured by a modified WCBS over time, for example, a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more change over time in fundus photographic appearance as measured by a modified WCBS from baseline at about 1-2, 2-4, 4-6, 8-10, 10-12, 12-14, 12-16, 16-18, 18-20, 20-22, 22-24 months, or at about 1, 3, 6, 9, 12, 15, 18, 21, or 24 months from baseline.

[0256] The efficacy of the methods provided herein can also be measured by a change over time in caregiver-reported visual outcomes measured over time using PedEyeQ, for example, a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more change over time in caregiver-reported visual outcomes assessed over time using PedEyeQ as measured by a modified WCBS from baseline at about 1-2, 2-4, 4-6, 8-10, 10-12, 12-14, 12-16, 16-18, 18-20, 20-22, 22-24 months, or at about 1, 3, 6, 9, 12, 15, 18, 21, or 24 months from baseline.

[0257] The efficacy of the methods provided herein can also be measured by a change over time in cognitively based adaptive behavior assessed over time using the VABs III and MSEL-Visual Reception, e.g., a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more change over time in cognitively based adaptive behavior assessed over time using the VABs III and MSEL-Visual Reception from baseline at about 1-2, 2-4, 4-6, 8-10, 10-12, 12-14, 12-16, 16-18, 18-20, 20-22, 22-24 months, or at about 1, 3, 6, 9, 12, 15, 18, 21, or 24 months from baseline.

[0258] The efficacy of the methods provided herein can also be measured by a change over time in the Hamburg Motor Scale score using the treated eye and the control eye, for example, a 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more change over time in the Hamburg Motor Scale score using the treated eye and the control eye as measured by the modified WCBS from baseline at about 1 to 2, 2 to 4, 4 to 6, 8 to 10, 10 to 12, 12 to 14, 12 to 16, 16 to 18, 18 to 20, 20 to 22, 22 to 24 months, or at about 1, 3, 6, 9, 12, 15, 18, 21, or 24 months.

[0259] In another aspect, vector shedding can be determined by measuring vector DNA in biological fluids such as tears, serum, or urine, for example, using quantitative polymerase chain reaction. In some embodiments, no vector gene copies are detectable in biological fluids (e.g., tears, serum, or urine) at any time point after vector administration. In some embodiments, fewer than 1,000, fewer than 500, fewer than 100, fewer than 50, or fewer than 10 vector gene copies / 5 μL are detectable by quantitative polymerase chain reaction in biological fluids (e.g., tears, serum, or urine) at any time point after administration. In certain embodiments, 210 vector gene copies / 5 μL or less are detectable in serum. In some embodiments, fewer than 1000, fewer than 500, fewer than 100, fewer than 50, or fewer than 10 vector gene copies / 5 μL are detectable by quantitative polymerase chain reaction in biological fluids (e.g., tears, serum, or urine) by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 weeks after administration. In certain embodiments, no vector gene copies are detectable in serum by 14 weeks after administration of the vector.

[0260] In another embodiment, the expression level of a transgene (e.g., a transgene encoded by Construct I, or TPP1) can be monitored by measuring the transgene product in the serum and / or CSF of a subject to which Construct I has been administered. The expression level of a transgene product (e.g., TPP1) can also be monitored in the eye (e.g., aqueous humor and / or vitreous humor) of a subject to which Construct I has been administered. Transgene expression can be measured by any suitable assay known in the art, including, but not limited to, Western blotting, electrochemiluminescence (ECL) immunoassay implemented using the Mesoscale Discovery (MSD) platform, and ELISA.

[0261] In some embodiments, the expression level of TPP1 in the eye may vary between different regions of the eye, for example, high levels of vector DNA may be detected in the retina and choroid / RPE (temporal, by injection) in the region of the bleb, as well as in the superior, inferior, and nasal quadrants, while low levels may be detected in the optic nerve, optic chiasm, and occipital lobe. In some embodiments, the rAAV provided herein (e.g., Construct I) exhibits restricted anterograde tropism along the visual pathway.

[0262] In some embodiments, administration of an rAAV provided herein (e.g., Construct I) to a subject results in a detectable level of TPP1 expression in the vitreous humor and / or aqueous humor of the subject's eye within about 3 months, about 6 months, about 9 months, about 12 months, about 15 months, about 18 months, about 21 months, or about 24 months of administration of the rAAV to the subject, and prior to administration of the rAAV, the level of TPP1 expression in the vitreous humor and / or aqueous humor was undetectable. In some embodiments, administration of a rAAV provided herein (e.g., Construct I) to a subject results in a detectable level of TPP1 expression in the vitreous humor and / or aqueous humor of the subject's eye within about 3 months, about 6 months, about 9 months, about 12 months, about 15 months, about 18 months, about 21 months, or about 24 months of administration of the rAAV to the subject, wherein prior to administration of the rAAV, the level of TPP1 expression in the vitreous humor and / or aqueous humor is undetectable, and the level of TPP1 expression in the subject's serum remains undetectable (e.g., undetectable using methods described herein or known in the art). In some embodiments, TPP1 is detectable or undetectable using an immunofluorescence assay. In some embodiments, TPP1 is detectable or undetectable using an immunostaining assay.

[0263] In some embodiments, administration of a rAAV provided herein (e.g., Construct I) to a subject results in an increase in TPP1 expression level (e.g., about 100-fold to about 150-fold increase) in the vitreous and / or aqueous humor of the subject's eye within about 3 months, about 6 months, about 9 months, about 12 months, about 15 months, about 18 months, about 21 months, or about 24 months of administration of the rAAV to the subject, compared to the TPP1 expression level before administration of the rAAV. about 500-fold, about 500-fold to about 1000-fold, about 1000-fold to about 1500-fold, about 1500-fold to about 2000-fold, about 2000-fold to about 2500-fold, about 2500-fold to about 3000-fold, about 3000-fold to about 4000-fold, about 4000-fold to about 5000-fold, about 5000-fold to about 10,000-fold, about 10,000-fold to about 15,000-fold, about 15,000-fold to about 20,000-fold, or more than about 20,000-fold increase). In some embodiments, administration of an rAAV provided herein (e.g., Construct I) to a subject results in an increase in TPP1 expression level (e.g., about 100-fold to about 500-fold, about 500-fold to about 1000-fold, about 1000-fold to about 1500-fold, about 1000-fold to about 1500-fold, about 1000-fold to about 1500-fold, about 1000-fold to about 1500-fold, about 1000-fold to about 1500-fold, about 1500-fold to about 2000-fold, about 2000-fold to about 21000-fold, about 21 ... The TPP1 expression level in the subject's serum remains approximately constant (e.g., within about 10%) compared to the pre-administration TPP1 expression level. In some embodiments, TPP1 expression is measured using an immunofluorescence assay. In some embodiments, TPP1 expression is detectable or undetectable using an immunostaining assay.

[0264] In some embodiments, administration of an rAAV provided herein (e.g., Construct I) to a subject results in TPP1 expression in the subject's retina, e.g., expression in, or a proportion of, the retinal pigment epithelium and / or photoreceptor outer segments, or expression throughout the entire depth of the retina. In some embodiments, TPP1 expression may be restricted to neurons.

[0265] Transgene product levels can be measured in patient samples of vitreous and / or aqueous humor from the anterior chamber of the treated eye. Alternatively, vitreous humor concentrations can be estimated and / or monitored by measuring the patient's serum concentration of the transgene product—the ratio of systemic to vitreous exposure to the transgene product is approximately 1:90,000. (See, e.g., the aqueous humor and serum concentrations of ranibizumab reported in Xu L, et al., 2013, Invest. Opthal. Vis. Sci. 54:1621, pp. 1616-1624 and Table 5, p. 1623, incorporated herein by reference in their entireties.)

[0266] Immunogenicity to Construct I can be assessed as anti-transgene product antibodies (aqueous humor, serum, and CSF), anti-AAV9 antibodies (serum, CSF), and T cell reactivity to AAV9 and Construct I transgene products via enzyme-linked immunospot (ELISPOT; whole blood).

[0267] In some embodiments, disease progression may be assessed by administration of CLN2 CRS-MX to pediatric patients. In some embodiments, disease progression may be assessed by administration of CLN2 CRS-MX to adult patients. In some embodiments, provided herein are methods of treating ocular symptoms associated with CLN2 Batten disease in a subject in need thereof, the method comprising administering to the eye of the subject a recombinant adeno-associated virus (rAAV) comprising a capsid and a vector genome, wherein the rAAV is AAV9, and the vector genome is AAV5' inverted terminal repeat (ITR), The promoter and a CLN2 coding sequence encoding the human tripeptidyl peptidase 1 (TPP1) protein; and AAV3'ITR, The method further includes monitoring the patient for a change, or lack thereof, in their CLN2 CRS-MX rating during and / or after administration of the vector. In some embodiments, the subject has a change from baseline in their CLN2 CRS-MX rating of +1 point, +2 points, +3 points, +4 points, +5 points, or +6 points. In some embodiments, the method delays or halts the progression of ocular symptoms associated with CLN2 Batten disease in the subject, as determined by a delayed reduction and / or maintenance of the subject's CLN2 CRS-MX rating over a period of 1 month or more, 2 months or more, 3 months or more, 6 months or more, 1 year or more, or 2 years or more.

[0268] In some embodiments, disease progression can be assessed by administering CLN2 CRS-LX to pediatric patients. In some embodiments, provided herein are methods of treating ocular symptoms associated with CLN2 Batten disease in a subject in need thereof, the method comprising administering to the eye of the subject a recombinant adeno-associated virus (rAAV) comprising a capsid and a vector genome, wherein the rAAV is AAV9, and the vector genome is AAV5' inverted terminal repeat (ITR), The promoter and a CLN2 coding sequence encoding the human tripeptidyl peptidase 1 (TPP1) protein; and AAV3'ITR, The method further includes monitoring the patient for a change, or lack thereof, in their CLN2 CRS-LX rating during and / or after administration of the vector. In some embodiments, the subject has a change from baseline in their CLN2 CRS-LX rating of +1 point, +2 points, +3 points, +4 points, +5 points, or +6 points. In some embodiments, the method delays or halts the progression of ocular symptoms associated with CLN2 Batten disease in the subject, as determined by a delayed reduction and / or maintenance of the subject's CLN2 CRS-LX rating over a period of 1 month or more, 2 months or more, 3 months or more, 6 months or more, 1 year or more, or 2 years or more.

[0269] 4.4 Treatment Systems, Devices, or Apparatus for Use in the Treatment Methods Described Herein Also provided herein are treatment systems, devices, and apparatuses for use in the treatment methods described herein, which may include one or more of the following: a bottle, a tube, a light source, a microinjector, and a foot pedal. In certain embodiments, the light source is a self-illuminating contact lens, which can be used to deposit the vector in the posterior segment of the eye, particularly to avoid damage to the optic disc, fovea, and / or macula (see, e.g., Chalam et al., 2004, Ophthalmic surgery and lasers. 35:76-77, incorporated herein by reference in its entirety). In certain embodiments, a self-illuminating contact lens is utilized during peri-injection to visualize the subretinal injection (see, e.g., Chalam et al., 2004, Ophthalmic surgery and lasers. 35:76-77, incorporated herein by reference in its entirety). In certain embodiments, a fiber optic chandelier is utilized via a second trocar to visualize the subretinal injection.

[0270] 4.5 Combination therapy The methods provided herein may be combined with one or more additional therapies.

[0271] The additional therapy may be administered before, concurrently with, or after the gene therapy treatment. In some embodiments, the additional therapy is enzyme replacement therapy (ERT) with recombinant TPP1 (e.g., Brineura® cerliponase alfa). 5. Arrays [Table 2] TIFF2023544803000004.tif224165TIFF2023544803000005.tif240165TIFF2023544803000006.tif127165 TIFF2023544803000007.tif228165TIFF2023544803000008.tif219165TIFF2023544803000009.tif237165 TIFF2023544803000010.tif220165TIFF2023544803000011.tif189165TIFF2023544803000012.tif223165 TIFF2023544803000013.tif225165TIFF2023544803000014.tif235165TIFF2023544803000015.tif132165 [Example]

[0272] 6. Working Example The examples in this section (ie, Section 6) are provided by way of illustration, not limitation.

[0273] 6.1 Example 1: Protocol for treating human subjects and assessing the efficacy of treatment with Construct I This example provides an exemplary protocol for treatment of human subjects to allow for evaluation of the efficacy of Construct I treatment. Construct I is administered at a dose of about or at least about 2 x 10 10 GC / eye or about or at least about 6 x 10 10 Construct I may be administered at a dose of approximately 1 x 10 GC / eye. 11 GC / mL ~ approx. 3.0×1011 It may be administered subretinal in a volume of 200 μL at a concentration of GC / mL.

[0274] 6.1.1 Patient population Patients treated according to the methods described in this example have biallelic CLN2 mutations and reduced leukocyte TPP1 activity. Patients also exhibit clinical signs or symptoms consistent with CLN2 disease (e.g., developmental delay, developmental decline, seizures, vision loss, or other symptoms) or have an older sibling with a confirmed CLN2 diagnosis.

[0275] Patients are receiving intraventricular Brineura enzyme replacement therapy. Patients may also have a baseline CRT of ≤210 μm in both eyes and a baseline CRT of ≥140 μm, and be up to 84 months of age. Alternatively, patients may be presymptomatic, have a baseline CRT of >210 μm in both eyes, and be 12 to 48 months of age. If CLN2 disease is diagnosed in an asymptomatic patient due to a family history of CLN2 disease in a sibling, the onset of vision loss in the affected sibling must be 84 months of age or younger. Patients may also have a baseline CRT of <140 μm in both eyes and a Weill Cornell Ophthalmology Severity Score <5 (no age restriction).

[0276] Patients have measurable visual acuity (VA) with an OKN-based VA test.

[0277] Patients may receive prednisolone (1 mg / kg / day for 7 days (i.e., days -3 to 4), up to a maximum dose of 40 mg / day) concurrently with Construct I treatment. After 7 days (3 days pre-op, day of surgery, 3 days post-op), patients may receive a reduced dose of 0.5 mg / kg / day, up to a maximum dose of 20 mg / day for an additional 10 days (i.e., days 5 to 14).

[0278] Patients may be receiving intraventricular Brineura enzyme replacement therapy every 2 weeks for the treatment of CLN2 Batten disease.

[0279] 6.1.2 Efficacy Endpoints The primary efficacy endpoint is the change from baseline in CRT measured by SD-OCT at day 360. Other efficacy endpoints include: AAV9 detected by PCR in serum and urine Change from baseline in VA as measured by OKN at day 360 (or longer), Change from baseline in central 1 mm ONL thickness as measured by SD-OCT at day 360 (or longer), Changes from baseline in measurements and appearance of retinal layers over time, as measured by SD-OCT at 360 days; Change from baseline in pupillary light reflex as measured by serial pupillometry at 360 days; Change from baseline in macular thickness / volume as measured by SD-OCT at day 360 Time to the accelerated decline phase of retinal degeneration, defined as reaching a CRT of 210 μm, and time from a CRT ≤ 210 μm to a loss of 30 μm. Change from baseline in CRT as measured by SD-OCT at day 360; SD-OCT anatomical markers, e.g., parafoveal ellipsoid zone, ELM, RNFL, Change from baseline in fundus appearance on fundus photographs over time as measured by a modified WCBS; Change from baseline in FAF at day 360, time to end-stage disease defined as CRT ≤ 110 μm, Change from baseline over time in caregiver-reported visual outcomes assessed using PedEyeQ, Change from baseline over time in cognitively based adaptive behaviors as assessed using the VAB III and MSEL-Visual Reception. Change from baseline in Hamburg Scale motor function score using the treated and control eyes, Change from baseline in the modified Hamburg Scale motor function score using the treated and control eyes.

[0280] 6.1.3 Pharmacodynamic endpoints Levels of TPP1 in aqueous humor, serum, and CSF are also assessed. AAV9 concentrations are measured in CSF. Antibodies to AAV9 may be measured in serum and CSF, and antibodies to the transgene product of Construct I (TPP1) may be measured in serum, CSF, and aqueous humor. T cell reactivity to AAV9 and Construct I TP is tested in PBMCs using ELISPOT with whole blood.

[0281] 6.1.4 Effectiveness Evaluation The efficacy of treatment with Construct I can be assessed using various assays. For example, retinal imaging can be performed using spectral-domain optical coherence tomography (SD-OCT) (macula and optic disc) using a Heidelberg Spectralis modular imaging system.

[0282] Axial length of the treated and control eyes can be measured using A-scan ultrasonography. Visual acuity by optokinetic nystagmus testing (OKN testing) may also be performed.

[0283] Fundus autofluorescence of the study eye can be performed using a Heidelberg Spectralis OCT instrument with a Flex Module.

[0284] Multicolor fundus imaging of the study eye may be performed using a Heidelberg Spectralis OCT instrument with a Flex Module.

[0285] A pupillary light reflex with pupillometry can be performed.

[0286] The PedEyeQ can be used to assess the effects of childhood eye disease on eye-related quality of life and functional vision in children up to 17 years of age (Hatt et al., 2019, Am J Ophthalmol. 200:201-17). PedEyeQ is available in multiple versions for children, surrogates, and parents, and can be tailored for age groups 0-4 and 5-11 years. Each age-specific proxy questionnaire consists of three general domains (functional vision, eye / vision distress, and social) and two domains (frustration / worry, and eye care) for patients aged 5 years and older only. Each domain consists of 5-10 items. Caregivers can select "never," "sometimes," or "always" as a response to each item, corresponding to a score of 2, 1, or 0, respectively. Each domain can be independently scored by the average Rasch-calibrated value of all completed items in the domain. Domain scores can be converted to a scale of 0 to 100, with 0 being considered the worst possible and 100 being considered the best possible measure of quality of life or functional vision.

[0287] The Vineland Adaptive Behavior Scales (VABS)-III (Sparrow et al., 2016, Vineland Adaptive Behavior Scales. 3rd ed. Bloomington, MN: Pearson) can be used to assess neurocognitive-based adaptive behavior in individuals from infancy through age 90. Four domains can be assessed, suitable for children under 7 years of age: communication, daily living skills, socialization, and motor skills. Items in each domain are scored from 2 to 0 based on the frequency with which the individual exhibits each adaptive skill / behavior, with 2 corresponding to almost always and 0 corresponding to never. Individual items are tallied and then calculated as a composite score that is compared to a standard age-adapted bell curve with a mean of 100 and a standard deviation of 15. Depending on which percentile a child's score falls in, their overall adaptive behavioral competence can be scored as low, moderately low, moderate, moderately high, or high.

[0288] The Mullen Early Learning Scales (MSEL) is a standardized assessment commonly used as a measure of cognitive development (Mullen, 1995, Scales of Early Learning (AGS ed.). Circle Pines, MN: American Guidance Service Inc.). The MSEL consists of five subscales measuring (a) gross motor, (b) fine motor, (c) visual reception (or nonverbal problem solving), (d) receptive language, and (e) expressive language skills. Each subscale can be standardized to calculate standard scores, percentiles, and age-equivalent scores.

[0289] The MSEL was adapted for use in this study as a clinical outcome assessment to measure the potential effects of treatment on visual processing and problem solving (i.e., MSEL-Visual Reception). In addition to the standard visual reception subscale, a modified version of the visual reception domain (first seven items) may be assessed, i.e., focusing on fixation and tracking while minimizing reliance on cognitive and fine motor dexterity. Examples include fixating and tracking a triangle, tracking a moving bull's-eye 180 degrees, and finding a dropped spoon.

[0290] The CLN2 Clinical Rating Scale (CRS) was developed specifically for CLN2 disease to assess changes in an individual's motor function, language, seizures, and vision over time. The original 12-point Hamburg Scale (Steinfeld et al., 2002, Am J Med Genet. 112(4):347-54) was developed to assess patients both retrospectively and prospectively. It has been used to collect the largest longitudinal natural history data (Nickel et al., 2018, Lancet Child Adolesc Health. 2(8):582-590. doi:10.1016 / S2352-4642(18)30179-2. Epub 2018 Jul 2). In 2018, updates to the motor and language domains of the scale were published (Wyrwich et al., 2018, J Inborn Errors Metab Screen. 6:1-7), providing more detailed definitions for the scale wording of the motor and language scores for use in prospective data collection in clinical trials. The motor and language domains together form a score ranging from 0 to 6 points. The highest score for each domain is 3 points, corresponding to normal or maximal ability, while a score of 0 indicates no remaining ability in that domain. These scales have previously been used in clinical studies, such as the 6-point motor-language scale used to evaluate the clinical effectiveness of Brineura (Schulz et al., 2018, N Engl J Med. 378(20):1898-1907). Motor function scales can be assessed using both the original Hamburg scale and the 2018 revised version. Patients are required to complete each test twice, once with each eye occluded, so that OD (right eye) and OS (left eye) scores are recorded. In total, four scores can be recorded for the two scales.

[0291] 6.1.5 Safety Assessment The safety of administration of Construct I can be assessed, for example, by ophthalmologic examination, physical examination vital sign assessment, or clinical safety laboratory assessment (including, for example, serum chemistry, hematology measurements, and urinalysis).

[0292] 6.1.6 Vector shedding Vector transgenes have the potential to spread to unintended recipients through shedding (release of the vector without infecting target cells and being cleared from the body via feces or body fluids), mobilization (transgene replication and migration from target cells), or germline transmission (gene transmission to offspring via semen). Vector shedding can be determined, for example, by measuring vector DNA in biological fluids such as tears, serum, or urine using quantitative polymerase chain reaction.

[0293] Blood (serum), urine, and tear sampling may be performed on Construct I patients for measurement of AAV9 vector concentrations.

[0294] Shedding data collected in these biofluids can provide a shedding profile of Construct I in the target patient population and can be used to estimate the likelihood of transmission to untreated individuals. Shedding can be measured using quantitative polymerase chain reaction (PCR).

[0295] 6.2 Example 2: Subretinal injection of Construct I into cynomolgus monkeys results in supraphysiological levels of TPP1 in the eye In late-stage infantile neuronal ceroid lipofuscinosis (CLN2) disease, mutations in the CLN2 gene lead to a lack of TPP1 enzyme activity, resulting in severe neurodegeneration within the central nervous system and retina. There are currently no approved therapies to treat the ocular manifestations of CLN2 disease, and visual loss represents a significant unmet need. The purpose of this study was to administer Construct I via subretinal injection to produce sufficient levels of TPP1 enzyme to prevent the ocular manifestations of CLN2 disease. Because mouse models of CLN2 disease do not exhibit overt retinal pathology, the pharmacodynamics, biodistribution, immunogenicity, and toxicity of Construct I were evaluated in cynomolgus monkeys. Groups of cynomolgus monkeys (4–5 / group) were administered Construct I at doses of ≥1 × 10 10The transgene was administered via subretinal injection (100 μL) at a dose of 100 μg / eye, and transgene expression (TPP1 concentration) and biodistribution in serum, aqueous humor, and vitreous humor were assessed for up to 13 weeks. Increased TPP1 concentrations were observed in the aqueous humor and vitreous humor, but not in serum. Transgene expression persisted for 3 months, although TPP1 concentrations partially decreased in some eyes due to the development of serum anti-TPP1 antibodies. In the eyes, high levels of vector DNA were observed in the retina and choroid / RPE in both bleb (temporal) and non-bleb (superior, inferior, and nasal quadrants) regions. Vector DNA was also present at low levels in the optic nerve, optic chiasm, and occipital lobe, but not in other brain regions, suggesting anterograde tropism. Immunostaining demonstrated that TPP1 protein was present in a wide range of retinal regions, primarily within photoreceptors, but also to a lesser extent in other cell layers. 1 × 10 10 In GC / eyes, TPP1 concentrations were 150-fold (aqueous humor) and 200-fold (vitreous humor) higher than in untreated animals, demonstrating that subretinal injection of Construct I can provide sufficient levels of TPP1 to attenuate the retinal degeneration seen in CLN2 patients.

[0296] 6.3 Example 3: Subretinal injection of Construct I into cynomolgus monkeys results in supraphysiological levels of TPP1 in the eye The aim of this study was to evaluate the levels of human TPP1 in the retina of cynomolgus monkeys after subretinal injection of Construct I, a recombinant adeno-associated virus of serotype 9 (AAV9) containing a human CLN2 (hCLN2) expression cassette.

[0297] 6.3.1 Method Groups of young cynomolgus monkeys (n = 5 / group, approximately 2-3 years old) received a single subretinal injection (100 μL) of Construct I at 0 (vehicle) or >1 × 10 10AAV9 was administered to both eyes at a dose of 1000 mg / eye. Animals were euthanized either 4 weeks (2 / group) or 13 weeks (3 / group) after administration. The study included assessment of TPP1 concentrations in aqueous humor, vitreous humor (terminal samples), and serum, immunogenicity (anti-AAV9 neutralizing antibodies [NAb] and anti-TPP1 antibodies) in serum, biodistribution (vector DNA), and TPP1 immunostaining.

[0298] TPP1 concentrations were determined by electrochemiluminescence (ECL) immunoassay implemented using the Mesoscale Discovery (MSD) platform.

[0299] 6.3.2 Results Some Construct I-treated animals were NAb positive before administration, and administration of Construct I had no significant effect on pharmacodynamics or biodistribution.

[0300] Subretinal injection of Construct I into healthy cynomolgus monkey eyes resulted in increased TPP1 concentrations in the aqueous humor over the course of the study, as well as in the vitreous humor (terminal samples only) after 3 months (Figures 4A and 4B). This was not reflected in serum, which remained comparable to control animals throughout the study (Figure 5). Terminal samples from animals treated with Construct I showed an increased incidence of anti-TPP1 antibodies, which may have resulted in decreased aqueous TPP1 concentrations in some animals.

[0301] High levels of vector DNA were detected in the retina and choroid / RPE in the region of the bleb (temporal) and in the superior, inferior, and nasal quadrants (FIGS. 6A and 6B).

[0302] Vector DNA is ≥ 1 × 10 11 In GC / eye, it was detected at low levels in the optic nerve, optic chiasm, and occipital lobe, suggesting a limited anterograde tropism along the visual pathway (FIGS. 7A and 7B).

[0303] TPP1 immunoreactivity was observed within widespread areas of the retina. 10In GC / eyes, strong TPP1 immunoreactivity was present only in the retinal pigment epithelium, parts of the photoreceptor outer segments, and occasional labeling of horizontal or amacrine cells (1 × 10 11 (GC / visually more obvious). 1×10 12 In contrast, strong TPP1 immunoreactivity was observed throughout the depth of the retina in GC / eyes (Fig. 8). 12 In GC / eyes, TPP1 immunostaining appeared to label all types of neurons present, including retinal ganglion cells and a much higher proportion of photoreceptor cells than in either of the other two doses. Regardless of dose, TPP1 immunoreactivity within the retina appeared restricted to neurons without obvious staining of Müller glia, even adjacent to where the subretinal vector deposits were located.

[0304] The percent area of ​​TPP1 stained retina was greater in animals treated with Construct I than that present in vehicle-treated animals (FIG. 9).

[0305] 6.3.3 Conclusion: 1×10 10 The GC / eye dose resulted in TPP1 concentrations that were approximately 170-fold (aqueous humor) and 200-fold (vitreous humor) higher than those in untreated cynomolgus monkeys.

[0306] Immunostaining revealed strong TPP1 immunoreactivity within the retinal pigment epithelium and in the outer segments of a proportion of photoreceptors, with 1 × 10 10 GC / eye with only occasional labeling of presumed horizontal or amacrine cells.

[0307] Data generated in cynomolgus monkeys demonstrate that subretinal injection of Construct I can provide sufficient levels of TPP1 to attenuate the retinal degeneration seen in CLN2 patients.

[0308] 6.4 Example 4: Subretinal injection of Construct I in cynomolgus monkeys leads to favorable safety levels The purpose of this study was to evaluate the safety of Construct I, a recombinant adeno-associated virus of serotype 9 (AAV9) containing a human CLN2 (hCLN2) expression cassette, through a 3-month toxicity study in cynomolgus monkeys. In addition to standard toxicity study endpoints, this study included evaluation of ocular physiological changes. Juvenile, reproductive, and genotoxicity were also discussed given the high unmet clinical need in children with CLN2 disease.

[0309] 6.4.1 Method A group of cynomolgus monkeys was given 1 × 10 10 ~1×10 13 Construct I was administered by subretinal injection at doses ranging from GC / eye. Tripeptidyl peptidase 1 (TPP1) concentrations in serum, aqueous humor, and vitreous humor, as well as biodistribution, were assessed to confirm transgene expression. Eyes under study were examined by direct and indirect ophthalmoscopy, OCT, electroretinogram, fundus photography, and histopathology. Efficacy was assessed over a period of up to 3 months. Young cynomolgus monkeys aged 2-3 years were included in the study.

[0310] 6.4.2 Results Subretinal injection of Construct I into cynomolgus monkeys showed elevated and sustained levels of TPP1 biodistribution, indicating normal transgene expression in this study.

[0311] The result is 1 x 10 10 The GC / eye dose was suggested to be the no-observed-adverse-effect level (NOAEL) for this study. Adverse effects or findings included ocular inflammation, OCT and fundus photography findings, and decreased visual function.

[0312] 1 x 10 to male and / or female cynomolgus monkeys 10 , 1×10 11、 1×10 12 , or 1 × 10 13 Subretinal administration of GC / eye construct I was performed at a dose of ≥ 1 × 10 11 GC / 1×10 11 or 1×10 12At the GC / eye dose, aqueous cells, vitreous opacity, or ocular inflammation observed in vitreous cells was less severe than that observed at the highest dose. Also, at lower doses (e.g., 1 × 10 11 or 1×10 12 The ocular inflammation observed in GC / eye responded well to systemic and / or topical anti-inflammatory therapy.

[0313] 1×10 10 Occasional vitreous cell inflammation observed in GC / eye was not considered an adverse effect. 10 In GC / eyes, occasional vitreous cell inflammation decreased in severity by day 22, but persisted in some eyes for 3 months. One animal showed more severe inflammation with severe aqueous cells, moderately severe flare, and moderately severe vitreous opacification. However, this was observed in one eye only on day 15, and topical anti-inflammatory therapy was applied. 1 × 10 10 No vector-related effects on fundus color photography, ERG, or visual evoked potentials were observed at the GC / eye dose, demonstrating that vitreous cell inflammation did not correlate with effects on visual function, even though minimal mononuclear cell infiltration was observed in one eye at the end of the study upon microscopic evaluation. This was consistent with the 1 × 10 10 These results suggest that occasional vitreous cell inflammation in GC / eyes does not affect visual function and can be controlled within mild levels.

[0314] No neurobehavioral, respiratory, or microscopic (cardiovascular) changes were observed in cynomolgus monkeys throughout the 3-month toxicity study.

[0315] 1×10 10 There were no findings suggestive of effects on the reproductive system at the GC / ocular dose. 10 It was not detected in the testes or ovaries of any animals up to the dose of GC / eye. In contrast, vector DNA was detected primarily in ocular tissues at days 29 and 92 under the same dose. This distinction was evident at doses of 1 x 10 10This supports the idea that GC / eye may be safe for the reproductive system due to the undetectable vector DNA levels therein.

[0316] Dose ≧1×10 11 The presence of vector DNA at very low copy numbers in GC / eye did not suggest germline effects. 11 GC was administered subretinaly to one male and 1.0 × 10 12 In one male administered GC / eye subretinally, vector DNA was detected in the testes at very low levels of 340 and 10 copies / μg DNA, respectively, on day 92. 12 In one female administered GC / eye into the suprachoroidal space (SCS), 344 copies / µg of DNA were detected in the ovary on day 29. No microscopic changes were observed in the testes or ovaries. The lack of such changes in the reproductive system suggests that very low DNA copies were present at doses ≥ 1 x 10 up to the highest dose in the study. 11 This suggests that even GC / eye could not be considered an adverse effect.

[0317] Construct I may pose a low risk of genotoxicity. Available scientific literature and clinical data report that AAV vector integration has not been observed (Wang et al., Adeno-associated virus vector as a platform for gene therapy delivery, Nature Reviews Drug Discovery, 2019, 18:358-378). Without the wild-type AAV integration machinery, Construct I cannot replicate in transduced cells. The recently approved AAV9, onasemnogene abeparvovec-xioi (ZOLGENSMA USPI, 2019), supports the low risk of insertional mutagenicity associated with recombinant adeno-associated virus (rAAV).

[0318] Immunotoxicity studies using Construct I can be evaluated by immune responses. In this study, humoral responses were monitored by detection of antibodies against the AAV9 capsid and antibodies directed against the transgene product, i.e., anti-TPP1 antibodies, in cynomolgus monkeys.

[0319] Juvenile toxicity of Construct I can be evaluated in 2- to 3-year-old cynomolgus monkeys. According to European Medicines Agency guidelines, this age range likely targets prepubertal monkeys, and therefore includes gestational age in the evaluation (EMEA / CHMP / SWP / 169215 / 2005).

[0320] 6.4.3 Conclusion 1×10 10 The dose level of GC / eye was, in this example, the NOAEL for subretinal administration of Construct I to cynomolgus monkeys. 10 The starting dose level of GC / eye may be implemented as the NOAEL. 1 x 10 11 or 1×10 12 The ocular inflammation observed with the GC / eye dose responded well to systemic and / or topical anti-inflammatory therapy.

[0321] Data generated from a 3-month toxicity study in cynomolgus monkeys demonstrate that subretinal administration of Construct I may provide a favorable safety profile. No neurobehavioral, respiratory, or microscopic changes were described following SR or SCS administration of Construct I. No specific juvenile, reproductive, or genotoxicity was observed.

[0322] 6.5 Example 5: Evaluation of Construct I in a Retina Chip Model of CLN2 Disease The goal of this study was to develop a physiologically relevant three-dimensional in vitro model of the human retina in CLN2 disease by combining human induced pluripotent stem cell (hiPSC) retinal organoids (ROs) with hiPSC-RPE in a retina-on-a-chip (RoC) model. This novel biomimetic platform allows for enhanced inner and outer segment formation and preservation, direct interaction between RPE and photoreceptors, and precisely controllable vasculature-like perfusion. This model provides support for the mechanism of action and potential benefits of Construct I, as well as characterizing disease phenotypes that are not possible in animal models or are not understood in children with CLN2.

[0323] 6.5.1 Method Dermal fibroblasts collected from a child prior to death from CLN2 disease were reprogrammed into human induced pluripotent stem cell lines and differentiated into RPE and retinal organoids (ROs) as described in Achberger et al. eLife 2019, 8, e46188 (incorporated herein by reference).

[0324] 6.5.2 Results Unlike control RPE, CLN2 RPE do not express TPP1. Restoration of TPP1 expression was demonstrated in CLN2 RPE 21 days after transduction with construct I (FIG. 10).

[0325] After RPE isolation, ROs were further matured. The photoreceptor marker recoverin was positive at day 200 of differentiation and absent at day 80. In control lines, TPP1 expression was detected primarily in the photoreceptor region in ROs at day 200, suggesting spatial and temporal specificity of TPP1 expression. Interestingly, ATP synthase subunit C (SCMAS), one of the accumulated substrates identified in CLN2, was present in CLN2 ROs only at day 200 in the photoreceptor region where TPP1 expression was detected in control ROs (Figure 11).

[0326] Treatment of CLN2 RO with Construct I on day 88, before detection of SCMAS accumulation, showed TPP1 expression along with preventative SCMAS accumulation, in contrast to untreated CLN2 RO, suggesting that early intervention can delay or prevent the accumulation of storage materials in RO (Figure 12).

[0327] 6.5.3 Conclusion Data generated in human CLN2 RPE using Construct I demonstrate that human CLN2 RPE and RO lack TPP1, and treatment with Construct I can restore TPP1 expression after treatment. Furthermore, when CLN2 RO are left untreated, there is an accumulation of SCMAS, a hallmark of CLN2 disease, which is prevented by pretreatment with Construct I. This data indicates that transduction of RO (including photoreceptors) with Construct I prevents the accumulation of lysosomal storage material associated with CLN2 degeneration.

[0328] 6.6 Example 6: Protocol for evaluation of Construct I in the Retinal Chip Model of CLN2 Disease The objective of this study was to evaluate the efficacy of Construct I, a recombinant adeno-associated virus of serotype 9 (AAV9) containing a human CLN2 (hCLN2) expression cassette, in treating ocular manifestations of CLN2 in human subjects using a retina-on-chip model.

[0329] 6.6.1 Differentiation of Patient and Control iPSCs into Retinal Organoids (RO) Four human induced pluripotent stem cell lines (iPSCs, two CLN2 patients) and two control lines were thawed and differentiated into retinal organoids (ROs) according to the retinal differentiation protocols described in Zhong et al. Nat. Commun. 2013, 5, 4047 and Achberger et al. eLife 2019, 8, e46188 (each incorporated herein by reference). ROs were cultured for a minimum of 3–6 months. Pigmented RPE cell clusters were harvested from differentiated ROs by manual dissection. These RPE cells were isolated and cultured in adherent culture. RPE cells from all available lines were cultured or frozen until use.

[0330] 6.6.2 Treatment of Retinal Organoids with Construct I Day 80 ROs of the available strains produced in section 6.6.1 are selected according to internal quality criteria (e.g., formation of segments, epithelial linear scars, absence of large necrotic areas).

[0331] Selected organoids are placed in a 96-well format and treated with Construct I. Three different concentrations are applied to available patient lines. Furthermore, one concentration of empty AAV is applied to one patient line to evaluate the specificity of the TPP1 gene expression effect. The effect of the treatment on the cells is monitored 5 weeks after the initial addition of Construct I. Endpoint methods include RNA analysis via qPCR (four biological replicates per condition) and protein analysis via immunocytochemistry (10 samples per condition). Organoids are stained for different retinal cell type markers, lysosomal markers (e.g., LAMP1 or LAMP2), TPP1 expression, and mitochondrial ATP synthase subunit C (SCMAS).

[0332] The above procedure is repeated on day 120 at RO.

[0333] 6.6.3 Treatment of Retinal Chips with Construct I For retina-on-a-chip (RoC) experiments, day 80 and day 120 RPE from all available lines are selected using the same criteria as in Section 6.6.2. RPE from available lines generated in Section 6.6.1 are selected according to internal quality criteria (passage, culture time, pigmentation, hexagonal cell shape, and general appearance). Three different concentrations of Construct I and a vehicle control are applied to available patient lines. The effect of treatment on RoC is monitored 5 weeks after the initial addition of Construct I. Endpoint methods include RNA analysis via qPCR and protein analysis via cryosectioning, followed by immunocytochemistry (8 samples per condition). RoC are stained for different retinal cell type markers, lysosomal markers (e.g., LAMP1 or LAMP2), TPP1 expression, and SCMAS.

[0334] 6.7 Example 7: Protocol for Treatment of Human RPE Organoids with Construct I and Investigating SCMAS Accumulation The purpose of this study was to treat human RPE organoids with Construct I and investigate SCMAS accumulation in RPE and retinal organoids by flow cytometry, single-cell RNA sequencing, and high-resolution confocal microscopy.

[0335] 6.7.1 Differentiation of Patient and Control iPSCs into Retinal Organoids (RO) Four human iPSC lines, two patient lines and two control lines, are thawed and differentiated into retinal organoids (ROs) as described in section 6.6.1. Pigmented RPE cell clumps are harvested from differentiated ROs by manual dissection. These RPE cells are cultured in suspension to form RPE organoids.

[0336] 6.7.2 Treatment of Retinal Pigment Epithelial Cell Organoids (RPEorg) with TPP1 Gene Therapy Our data suggest that adherent RPE cells cultured in monolayer for a limited period of time do not represent an accurate model for recapitulating disease phenotypes such as ATP-C deposits. On the other hand, RPE orgs, due to their 3D structure and long-term culture, have been shown to be a solid model for studying the effects of TPP1 mutations and ATP-C accumulation, making them an interesting platform for evaluating the effects of TPP1 gene therapy in RPE cells. To this end, RPE orgs at >150 days (phenotype onset) from the available lines generated in section 6.7.1 are selected according to internal quality criteria (e.g., uniform pigmentation, absence of non-RPE areas). The selected RPE orgs are then placed in a 96-well format, for example, and subjected to treatment with Construct I. The effects of the treatment on the cells are monitored 5 weeks after the initial addition of Construct I. Endpoint methods include RNA analysis via qPCR (four biological replicates per condition) and protein analysis via immunocytochemistry (10 samples per condition). Stain ROs for TPP1 expression, SCMAS, and lysosomal markers (e.g., LAMP1 or LAMP2).

[0337] 6.7.3 Analysis of TPP1 mutations and the effect of TPP1 gene therapy on RPEorg RPE cells from two control lines, two patient lines, and two patient lines treated with a concentration of TPP1 gene therapy (e.g., Construct I) at >150 days were selected, dissociated, and subjected to single-cell RNA sequencing (sc-RNAseq) 5 weeks after AAV treatment. The sc-RNAseq assessment determined the effect of TPP1 mutations on gene expression, including but not limited to SCMAS accumulation. Finally, the analysis identified the consequences of TPP1 gene therapy (e.g., Construct I) on RPE cell signaling, survival, and apoptosis.

[0338] 6.7.4 Cell-type specific analysis of TPP1 mutations and the effect of TPP1 gene therapy on RO ROs from two control lines, two patient lines, and two patient lines treated with a concentration of TPP1 gene therapy (e.g., Construct I) at day 120 were selected, dissociated, and subjected to single-cell RNA sequencing (sc-RNAseq) 5 weeks after AAV treatment. This analysis evaluates retinal cell types that express TPP1 (control lines) and cell types that are more efficiently infected by TPP1 gene therapy (patient lines). Furthermore, sc-RNAseq assessment determines the effect of TPP1 mutations on gene expression, including but not limited to SCMAS accumulation. Finally, the analysis identifies the consequences of TPP1 gene therapy on cell signaling, survival, and apoptosis.

[0339] 6.7.5 SCMAS accumulation survey To identify retinal cell types affected by SCMAS deposits and to highlight the correlation between TPP1 expression / absence and SCMAS, we analyzed ROs by flow cytometry (FC) after staining with specific antibodies for SCMAS, TPP1, and one for each retinal cell type (rods, cones, Müller glia, horizontal cells, amacrine cells, bipolar cells).

[0340] To this end, a protocol for Fc detection of these antibodies is first established. Then, three to five retinal cells from two control lines, two patient lines, and two patient lines treated with a certain concentration of TPP1 gene therapy (e.g., Construct I) at day 120 are selected, dissociated, and subjected to flow cytometry analysis after staining with specific antibodies for ATP-C, TPP1, and one for each retinal cell type.

[0341] Finally, retinal cell types showing SCMAS accumulation will be further analyzed by high-resolution confocal microscopy. To this end, three to five retinal cells from two control and two patient lines at >155 days will be stained for SCMAS and individual markers or retinal subtypes to assess SCMAS deposition morphology and cellular localization.

[0342] 6.8 Example 8: The CLN2 Clinical Rating Scale-Motor Extended (CLN2 CRS-MX) improves assessment of ambulatory function. In late-stage infantile neuronal ceroid lipofuscinosis 2 (CLN2) disease, rapid disease progression was originally quantified using the Hamburg CLN2 scale. The Hamburg motor domain was subsequently adapted into the CLN2 Clinical Rating Scale-Motor (CLN2 CRS-M) and used to quantify loss of motor function over the natural disease course compared with treatment response to intraventricular enzyme replacement therapy (ERT) with cerebellar septal ...

[0343] The aim of this study was to provide a broader and more granular measure, termed CLN2-CRS-MX, to assess the impact of CLN2 disease impairment on walking ability.

[0344] 6.8.1 Method Item derivation was based on identifying the primary CLN2 disease concept of interest.

[0345] Key concepts were identified from a targeted literature review, clinician-expert interviews, two virtual caregiver focus groups, and one year of ongoing biweekly meetings with six CLN2 clinician-experts.

[0346] Clinician interviews and caregiver focus groups discussed the main symptoms and impact of CLN2, particularly as it relates to motor function, differences in disease progression in ERT-treated and ERT-naive patients, and ways to improve the granularity of motor function assessment in CLN2 CRS to make it more useful for evaluating treatment effects in clinical trials.

[0347] The iterative development process included pilot applications and numerous item revisions. A tracking matrix was used to document the rationale for all scale iterations and changes.

[0348] Interrater reliability, i.e., the level of agreement between clinicians, was calculated as the percentage of agreement between the four raters.

[0349] Assessments were administered and scored by a primary clinician and also independently scored by an observer clinician.

[0350] Each assessment was videotaped and scored independently by two additional clinicians.

[0351] Thirty patients, aged 20 months to 16 years (mean 7.8 years), were individually scored on the CLN2 CRS-MX scale by four clinicians. All patients were currently receiving endorphin therapy (ERT).

[0352] 6.8.2 Results In this study, a detailed administration, scoring, and training manual for the CLN2 CRS-MX scale was developed.

[0353] In this study, a typical walking task was developed to determine symptom progression. Clinicians and caregivers generally characterized CLN2 disease by significant progression of symptoms that adversely affected gait. Trunk hypotonia, limb hypertonia, and ataxia were commonly reported symptoms affecting motor function. As symptoms worsened, patients reported an increasing need for assistance, e.g., grasping one hand, both hands, or the trunk for balance, until they were no longer able to walk. In this study, clinicians supported expanding the original CLN2 CRS-M 10-step walking task to a walking task that increased walking distance, considered the ability to safely stop, assessed changes in direction, and measured the level of assistance needed for walking.

[0354] Table 2 provides an overview of the rating criteria and a comparison with the original CLN2 CRS-M. Figure 2 shows an overview of the CLN2 CRS-MX scoring flow chart for determining mobility ratings. [Table 3] TIFF2023544803000017.tif140165

[0355] For interrater agreement, the level of agreement between clinicians for CLN2 CRS-MX is 1.0, indicating complete / perfect agreement (see Landis, JR, Koch, GG. An application of hierarchical kappa-type statistics in the assessment of majority agreement among multiple observers, Biometrics, 1977. Level of agreement range 0.81-0.99).

[0356] 6.8.3 Conclusion The CLN2 CRS-MX provided greater granularity, improved item relevance, and increased number of response options in measuring the impact of CLN2 disease on walking ability.

[0357] This scale was designed for children with CLN2, but may be relevant to other neuromuscular or neurodegenerative disorders that coexist with similar disorders.

[0358] 6.9 Example 9: The CLN2 Clinical Rating Scale-Language Extended (CLN2 CRS-LX) Improves Assessment of Language Function The loss of language function during the natural CLN2 disease course compared with the treatment response to ERT was quantified using the CLN2 Clinical Rating Scale-Language (CLN2 CRS-L). However, disease progression and phenotypic evolution are slow and imperceptible in children treated with ERT. The aim of this study was to provide a broader and more granular measure, termed the CLN2 CRS-LX, that captures changes in expressive and nonverbal communication skills.

[0359] 6.9.1 Method Item derivation was based on identifying the primary CLN2 disease concept of interest.

[0360] Key CLN2 disease concepts were identified from a targeted literature review [not specified in the materials], clinician-expert interviews, two virtual caregiver focus groups, and one year of ongoing biweekly meetings with six CLN2 clinician-experts.

[0361] Clinician interviews and caregiver focus groups discussed the main symptoms and impact of CLN2, particularly as it relates to language function, differences in disease progression in ERT-treated and ERT-naive patients, and ways to improve the granularity of language function assessment in CLN2 CRS to make it more useful for evaluating treatment effects in clinical trials.

[0362] The iterative development process included pilot applications and numerous item revisions. A tracking matrix was used to document the rationale for all scale iterations and changes.

[0363] Interrater reliability, i.e., the level of agreement between clinicians, was calculated as the percentage of agreement between the four raters.

[0364] Assessments were administered and scored by a primary clinician and also independently scored by an observer clinician.

[0365] Each assessment was videotaped and scored independently by two additional clinicians.

[0366] Thirty patients, aged 20 months to 16 years (mean 7.8 years), were individually scored on the CLN2 CRS-MX by four clinicians. All patients were currently receiving endorphin therapy (ERT).

[0367] 6.9.2 Results This study developed a detailed administration, scoring, and training manual for the CLN2 CRS-LX, including specific guidance on the use of prompts to determine expressive and nonverbal communication abilities.

[0368] The CLN2 CRS-LX measured children's expressive language abilities to communicate requests and needs, including babbling, vocabulary and phrase development, and unintelligible vocalizations and gestures. Clinicians and caregivers generally reported that children with CLN2 experience progressive language loss that impacts daily activities. They reported a peak vocabulary size of 20 to 100 words in the age range of 2 to 3.5 years, and that children with CLN2 used a range of communication strategies, including single words and two-word phrases, unintelligible vocalizations, and gestures. In this study, clinicians supported the development of an expanded CLN2 CRS for language that differentiates functional expectations by age, includes more response options, and takes into account vocabulary and phrase development, vocalizations, and gestures.

[0369] An overview of the rating criteria and a comparison with the original CLN2 CRS-L are provided in Tables 3 and 4. Scoring flowcharts were created for each CLN2 CRS-LX age category (1 to <2 years, 2 to <3 years, and ≥3 years). Figure 3 shows the flowchart for 2 to <3 years as an example. [Table 4] [Table 5] TIFF2023544803000020.tif134165

[0370] Interrater agreement showed that the level of agreement between clinicians for CLN2 CRS-LX was 0.933, indicating complete / perfect agreement (see Landis, JR, Koch, GG. An application of hierarchical kappa-type statistics in the assessment of majority agreement among multiple observers, Biometrics, 1977. Level of agreement range 0.81-0.99).

[0371] 6.9.3 Conclusion The CLN2 CRS-LX provided greater granularity, improved item relevance, and increased the number of response options in measuring the impact of CLN2 disease on expressive language and nonverbal communication abilities.

[0372] This scale was designed specifically for children with CLN2, but may be relevant to other neuromuscular or neurodegenerative disorders that coexist with similar disease disorders.

[0373] equivalent Although the present invention has been described in detail with reference to specific embodiments thereof, it will be understood that variations that are functionally equivalent are within the scope of the invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be within the scope of the appended claims. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the scope of the following claims.

[0374] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Claims

1. 1. A pharmaceutical composition for treating an ocular condition associated with CLN2 Batten disease in a subject in need thereof, comprising a recombinant adeno-associated virus (rAAV) vector, the treatment comprising administering to the eye of the subject 2×10 １０ Genome copies / eye or 6 x 10 １０ administering a genome copy / eye of said rAAV vector, wherein said rAAV vector comprises a capsid and a vector genome, said rAAV being AAV9, and said vector genome comprising: a. the AAV 5' inverted terminal repeat (ITR); b. a promoter; c. The CLN2 coding sequence encodes the human tripeptidyl peptidase 1 (TPP1) protein; d. AAV 3'ITR.

2. The pharmaceutical composition of claim 1, wherein the AAV 5' ITR and / or the AAV 3' ITR are derived from AAV2.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the coding sequence of (c) is a codon-optimized human CLN2 that is at least 70% identical to the native human coding sequence of SEQ ID NO:

2.

4. 3. The pharmaceutical composition of claim 1 or 2, wherein the coding sequence of (c) is SEQ ID NO:

3.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the promoter is a chicken beta actin promoter.

6. The pharmaceutical composition according to any one of claims 1 to 4, wherein the promoter is a hybrid promoter comprising a CBA promoter sequence and a cytomegalovirus enhancer element.

7. 7. The pharmaceutical composition of any one of claims 1 to 6, wherein the vector genome further comprises polyA, optionally wherein the polyA is synthetic or derived from bovine growth hormone (bGH), human growth hormone (hGH), SV40, rabbit β-globin (RGB), or modified RGB (mRGB).

8. 8. The pharmaceutical composition of any one of claims 1 to 7, wherein the vector genome further comprises an intron, optionally the intron is derived from CBA, human beta globin, IVS2, SV40, bGH, alpha-globulin, beta-globulin, collagen, ovalbumin, or p53.

9. 9. The pharmaceutical composition of any one of claims 1 to 8, wherein the vector genome further comprises an enhancer, optionally wherein the enhancer is a CMV enhancer, an RSV enhancer, an APB enhancer, an ABPS enhancer, an alpha mic / bik enhancer, a TTR enhancer, en34, or an ApoE.

10. As a result of said treatment, said subject: a. Central retinal thickness (CRT), measured by SD-OCT; b. Outer nuclear layer (ONL) thickness measured by SD-OCT; c. Pupillary light reflex, measured by pupillometry; d. Macular thickness / volume measured by SD-OCT; e. Total retinal thickness measured by SD-OCT; f. Retinal nerve fiber layer as measured by SD-OCT; g. Inner nuclear layer, as measured by SD-OCT; h. Photoreceptors (PR) + retinal pigment epithelium (RPE) as measured by SD-OCT; i. outer segment plus RPE (OS+RPE) as measured by SD-OCT; and / or j. Ellipsoid Zone (EZ), measured by SD-OCT The pharmaceutical composition of any one of claims 1 to 9, having a change from baseline in

11. As a result of said treatment, (a) the subject has a delayed onset of retinal deterioration compared to comparable clinical progression with standard of care; (b) the subject has a delayed onset of vision loss compared to a comparable clinical progression with standard treatment; (c) resulting in a detectable level of TPP1 expression in the vitreous and / or aqueous humor of the eye of the subject within 3 months of administration of the rAAV to the subject; or (d) the level of TPP1 expression in the serum of the subject remains undetectable.

12. The pharmaceutical composition of claim 11(c), wherein the level of TPP1 expression in the vitreous and / or aqueous humor was undetectable prior to administration of the rAAV.

13. The pharmaceutical composition of any one of claims 1 to 12, wherein the subject is concurrently receiving intraventricular Brineura® enzyme replacement therapy.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the subject is a human.

15. The pharmaceutical composition of any one of claims 1 to 14, wherein the administering step comprises administering the rAAV vector to the subretinal space of the eye.

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