Gene therapy for neuronal ceroid lipofuscinosis

Abstract

To provide a method and a composition for treating Batten disease.SOLUTION: A composition includes a recombinant adeno-associated virus (rAAV), the rAAV including AAV capsid, and a vector genome packaged therein, where the vector genome includes: (a) an AAV 5' inverted terminal repeat (ITR) sequence; (b) a promoter; (c) a CLN2 coding sequence encoding a human TPP1; and (d) an AAV 3'ITR. Further, a method for treating Batten disease includes administering the rAAV to a subject in need thereof by a plurality of routes.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] This application is a continuation of U.S. Provisional Application No. 62 / 767,410, filed November 14, 2018. and the benefit of U.S. Provisional Application No. 62 / 924,060, filed October 21, 2019. No. 60 / 699,999, filed on Dec. 1, 2003, and both applications are incorporated herein by reference in their entireties.

[0002] Reference to an electronically submitted sequence listing In this application, the file name "12656-125-228" created on November 3, 2019 _SEQ_LISTING", a text file with a file size of 36,393 bytes The sequence listing submitted herewith is incorporated by reference. [Background technology]

[0003] Neuronal ceroid lipofuscinosis (NCL) is a group of rare, inherited neurodegenerative disorders. They are characterized by autofluorescent lipoprotein pigments similar to the ceroid and lipofuscin found in patients. NCL is considered the most common neurogenetic storage disorder, involving accumulation of muscle abnormally increased tension or spasms, blindness or vision loss, dementia, impaired muscle coordination, intellectual disability It is associated with diverse but progressive symptoms, including disability, movement disorders, seizures, and unsteady gait. The disease occurs in approximately 1 in 12,500 people. There are three main types of NCL: There are types: adult (Koufus' or Parry's disease); juvenile and late-infantile (Janski's disease); Neuronal ceroid lipofuscinosis (NCL) is a condition that originally developed It was defined by age and clinical symptoms (as described herein). Reclassification based on new molecular findings has led to a more accurate diagnosis than previously suggested by the clinical phenotype. Evidence of much greater overlap was provided for different genetic variants.

[0004] At least 20 genes associated with NCL have been identified. NCL patients have a pepstatin-insensitive enzyme called tripeptidyl peptidase 1 (TTP1). TTP1 lacks the N-terminus of the polypeptide and the lysosomal peptidase. 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 infancy. However, later onset has been reported. Over 58 mutations have been described in CLN2. It has been done.

[0005] CLN2 disease, a variant of Batten disease, is a rare lysosomal storage disorder (LSD) that The incidence rate is estimated to be 0.07-0.51 per 100,000 live births (Augestad et al.,2006;Claussen et al.,1992;Mole et al. al.,2013;National Batten Disease Registry ry;Poupetova et al.,2010;Santorelli et a l., 2013; Teixeira et al., 2003). CLN2 disease is characterized by Located at 11q15, it encodes the soluble lysosomal enzyme tripeptidyl-peptidase-1 (TP A lethal autosomal recessive disorder caused by mutations in the CLN2 gene, which encodes P1 It is a chronic neurodegenerative LSD caused by mutations in the CLN2 gene and the subsequent lack of TPP1 enzyme activity. Loss of ATP leads to lysosomal accumulation of storage materials and neurodegeneration in the brain and retina (Liu et al. l., 1998; Wlodawer et al., 2003). CLN2 disease is characterized by a 2- It is characterized by early onset at age 4 years and is usually characterized initially by recurrent seizures (epilepsy) and movement disorders. The disorder also causes loss of previously acquired skills. Epilepsy is often resistant to medical treatment and can lead to mental retardation. A general decline in motor function occurs rapidly and uniformly between the ages of 3 and 5 (Schulz et al., 2013), followed by early death by mid-childhood (Nickel M et al., 2016; Worgall et al., 2007).

[0006] Recombinant TPP1 (Brineura® Seriponase α, BioMarin Enzyme replacement therapy (ERT) by Pharmacists is a promising treatment for CLN2 disease. It has recently been approved in the United States (US) and European Union (EU) for the treatment of It is administered as a biweekly infusion into the lateral ventricle via an implantable device. The clinical utility of Pharmacist (trademark) has been limited by the FDA to stabilizing motor function. However, the European Medicines Agency (EMA) has determined that language skills also have a positive effect ( Brineura®, FDA Drug Approval Review Summary; Brineura® European Published Pharmaceuticals Review Reports (EPARs); Schulz et al., 2016) Brineura® has the specialized expertise to implant ports directly into the brain. This requires a two-week period in a medical setting by trained professionals familiar with intracerebroventricular (ICV) administration. It should be administered during a 4-hour infusion every 4 hours. a® have short half-lives, estimated at 7 hours and 11.5 days, respectively. The main reason is the need for repeated injections (Brineura®, E Therefore, repeated administration of ERT imposes a heavy burden on patients and a high incidence of morbidity. However, persistent long-term TPP1 enzyme activity is observed in the central nervous system (CNS) of patients with CLN2 disease. There remains a significant unmet need for novel therapeutics that can provide essential activity. Therefore, it is possible to deliver TPP1 to a subject in need of treatment for CLN2 disease, There is a need for compositions useful for expressing canine TPP1 (rAAV2.caTPP1). A single administration of recombinant adeno-associated virus (rAAV) expressing TP primarily in ependymal cells High expression of P1 leads to secretion of the enzyme into the cerebrospinal fluid, which has been shown to be clinically useful. Katz et al., Sci Transl, incorporated herein by reference. Med. 2015 Nov 11;7(313):313ra180; and KATZ ,et al,Gene therapy 2017 Feb 24(4):215-2 23 However, AAV2 does not penetrate the brain parenchyma and targets neurons. Therefore, the expected utility is limited compared to that achievable with novel neurotropic AAVs. do. Summary of the Invention

[0007] Provided herein are aqueous suspensions suitable for administration to subjects with Batten disease. In one embodiment, the suspension comprises an aqueous suspension and about 7.5×100 ml of a soluble form ... 0 12 GC(7.5×10 9 GC / g brain) ~ 2.7 × 10 15 GC(2.1×1012 G C / g brain) containing recombinant adeno-associated virus (rAAV) virus particles, V has an AAV capsid and packages a vector genome containing: (a) AAV 5' inverted repeat (ITR) sequence; (b) promoter; (c) human (d) the CLN2 coding sequence encoding TPP1; and (e) the AAV 3'ITR.

[0008] Also herein, recombinant adeno-associated viruses (rAAVs) are referred to as vectors that are capable of expressing the virus via the first and second pathways. and administering to a subject in need thereof via a route The present invention provides a method for treating Barnett's disease, wherein the first pathway and the second pathway are mediated by central nervous system (CN) S), the first pathway is to a brain region, and the second pathway is to a spinal cord region. The recombinant adeno-associated virus (rAAV) is a pathway to the AAV capsid and and a vector genome packaged therein, the vector genome comprising: (a) AAV 5' inverted terminal repeat (ITR) sequence; (b) promoter; (c) human TP the CLN2 coding sequence encoding P1; and (d) the AAV 3'ITR.

[0009] In certain embodiments, the first route is intracerebroventricular (ICV) or intracisternal (IC). In certain embodiments, the second route is intrathecal (IT-L). In some embodiments, the method includes administering the rAAV to the subject via a third route. The third route may be intracerebroventricular (ICV), intracisternal (IC), lumbar intrathecal, or intracranial. Intravenous, intravascular, intraarterial, intramuscular, intraocular, subcutaneous, and intradermal administration is preferred.

[0010] In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises intracerebroventricular (ICV) ) and intrathecal (IT-L) routes to deliver the rAAV to subjects in need thereof. In certain embodiments, the method of treating CLN2 Batten disease in a subject includes co-administering The method involves the intracisternal (IC) and intrathecal lumbar (IT-L) routes of delivery of the rAAV. In certain embodiments, the method comprises co-administering the CLN2 antibody to a subject in need thereof. Treatment of Tutten's disease is available via intraventricular (ICV), intrathecal (IT-L), and intravenous routes. and co-administering the rAAV to a subject in need thereof via a cellular immunoglobulin (mAb). In this state, the method of treating CLN2 Batten disease in a subject includes intracisternal (IC), lumbar intrathecal (I) TL) and co-administering the rAAV to a subject in need thereof via the intravenous route. This includes:

[0011] In another embodiment, recombinant adeno-associated virus (rAAV) is prepared by the first and second pathways. administering to a subject in need thereof a CLN2 batten in a subject via The present invention provides a method for treating a disease, wherein the first pathway is a pathway to the central nervous system (CNS), The second route delivers rAAV outside the CNS and delivers the recombinant adeno-associated virus (rAAV) to the host. AAV (AAV) contains the AAV capsid and the vector genome packaged within it. The vector genome comprises: (a) an AAV 5' inverted terminal repeat (ITR) sequence; (b) (c) a CLN2 coding sequence encoding human TPP1; and (d) an AAV In certain embodiments, the first route is intrathecal (IT-L), intravenous, or intrathecal. In certain embodiments, the second route is intravenous (ICV) or intracisternal (IC). Intravenous, intravascular, intraarterial, intramuscular, intraocular, subcutaneous, and intradermal. In an embodiment, the second route is intravenous.

[0012] In another embodiment, recombinant adeno-associated virus (rAAV) is prepared by the first and second pathways. administering to a subject in need thereof a CLN2 batten in a subject via The present invention provides a method for treating a disease, wherein the first pathway is a pathway to the central nervous system (CNS), The second route delivers rAAV to the liver, and the recombinant adeno-associated virus (rAAV) , comprising an AAV capsid and a vector genome packaged therein, The genome includes: (a) the AAV 5' inverted terminal repeat (ITR) sequence; (b) the promoter sequence; (c) the CLN2 coding sequence encoding human TPP1; and (d) AAV 3′I In certain embodiments, the first route is intrathecal (IT-L), intraventricular (IC), or intrathecal. In certain embodiments, the second route is intravenous, intravascular (IV), or intracisternal (IC). In certain embodiments, the intravenous route is selected from the group consisting of intravenous, intraarterial, intramuscular, intraocular, subcutaneous, and intradermal. In this case, the second route is intravenous.

[0013] In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises intrathecal and intravenous and co-administering the rAAV to a subject in need thereof via an intravenous route. In embodiments, the method of treating CLN2 Batten disease in a subject comprises administering an intrathecal (IT-L) and co-administration of the rAAV to a subject in need thereof via an intravenous route. In certain embodiments, the method for treating CLN2 Batten disease in a subject comprises administering intraventricular (IV) and co-administration of the rAAV to a subject in need thereof via intravenous (IV) and intravenous routes. In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises administering intracisternal ( and co-administration of the rAAV to a subject in need thereof via intravenous (IC) and intravenous routes. Includes:

[0014] In certain embodiments, the methods for treating CLN2 Batten disease provided herein include treating the second and administering the rAAV via the first route simultaneously with administering the rAAV via the second route. The method may include administering AV.

[0015] In certain embodiments, the methods for treating CLN2 Batten disease provided herein include treating the second administering the rAAV via the first route before administering the rAAV via the second route In certain embodiments, the CLN2 Batten disease vectors provided herein may include administering the vector to a patient. The treatment method comprises administering the rAAV via the second route, followed by administration of the rAAV via the first route. The method may include administering the rAAV to a patient.

[0016] In certain embodiments, the methods for treating CLN2 Batten disease provided herein include administering to the subject In certain embodiments, the methods provided herein may result in increased TPP1 activity in the spinal cord of a subject. A method for treating CLN2 Batten disease may result in an increase in hepatic TPP1 activity in the subject. In certain embodiments, the methods of treating CLN2 Batten disease provided herein include administering to the subject In certain embodiments, the CL provided herein may result in an increase in serum TPP1 activity. A method for treating N2 Batten disease may result in a decrease in microglial activity within the cortex of the subject. In certain embodiments, the methods for treating CLN2 Batten disease provided herein include This may result in increased TPP1 activity in the elephant brain.

[0017] In certain embodiments, the rAAV is administered in a therapeutically effective amount.

[0018] In certain embodiments, the subject is a human.

[0019] In certain embodiments, the coding sequence of (c) is a codon-optimized human CLN2, is at least 70% identical to the native human coding sequence of SEQ ID NO: 2. In one embodiment, the coding sequence of (c) is SEQ ID NO:3.

[0020] In certain embodiments, the rAAV capsid is AAV9 or a variant thereof.

[0021] In a specific embodiment, the promoter is the chicken beta actin (CBA) promoter. In certain embodiments, the promoter comprises a CBA promoter sequence and a cytomegalovirus promoter sequence. It is a hybrid promoter containing a nucleotide enhancer element.

[0022] In certain embodiments, the AAV 5'ITR and / or the AAV 3'ITR are 2 is derived from

[0023] In certain embodiments, the vector genome further comprises polyA. The polyA may be synthetic polyA or may be derived from bovine growth hormone (bGH), human growth hormone (HGH), or other suitable polyA. (hGH), SV40, rabbit β-globin (RGB), or modified RGB (mRGB) This is the origin.

[0024] In certain embodiments, the vector genome further comprises an intron. Introns include CBA, human beta globin, IVS2, SV40, bGH, and alpha globin. These may be derived from leukocyte antigens, beta-globulin, collagen, ovalbumin, or p53.

[0025] In certain embodiments, the vector genome further comprises an enhancer. So, the enhancers are CMV enhancer, RSV enhancer, and APB enhancer. , ABPS enhancer, αmic / bik enhancer, TTR enhancer, en3 4. ApoE.

[0026] In certain embodiments, the vector genome is between about 3 kilobases and about 5.5 kilobases in size. In certain embodiments, the vector genome is about 4 kilobases in size.

[0027] In certain embodiments, suspension cell lines capable of producing rAAV are grown in suspension culture. In certain embodiments, the rAAV is produced using a method comprising: The cell line is a HEK293 suspension cell line.

[0028] Pharmaceutical compositions are also provided herein. In certain embodiments, and providing a pharmaceutical composition comprising: (a) recombinant adeno-associated virus (rAAV); (b) sodium chloride, (c) magnesium chloride, (d) potassium chloride, (e) dextrose, (f) Poloxamer 188, (g) monobasic sodium phosphate, and (h) dibasic sodium phosphate; In this case, the recombinant adeno-associated virus (rAAV) contains the AAV capsid and its The vector genome includes a vector genome packaged in a portion, the vector genome including: ) AAV 5' inverted repeat (ITR) sequence; (ii) promoter; (iii) human TPP 1; and (iv) the AAV 3′ ITR.

[0029] In certain embodiments, the pharmaceutical composition further comprises calcium chloride.

[0030] In certain embodiments, the sodium chloride, the magnesium chloride, the potassium chloride , the dextrose, the poloxamer 188, the monobasic sodium phosphate, the dibasic sodium phosphate The sodium phosphate and calcium chloride may be present in anhydrous, monohydrate, dihydrate, Trihydrate, tetrahydrate, pentahydrate, hexahydrate, heptahydrate, octahydrate, nonahydrate, or 10 hydrate It is in the form of a hydrate.

[0031] In certain embodiments, the pharmaceutical composition comprises: (a) the rAAV; (b) sodium chloride at a concentration of about 8.77 g / L; (c) magnesium chloride hexahydrate at a concentration of about 0.244 g / L; (d) potassium chloride at a concentration of about 0.224 g / L; (e) calcium chloride dihydrate at a concentration of about 0.206 g / L; (f) anhydrous dextrose at a concentration of about 0.793 g / L; (g) poloxamer 188 at a concentration of about 0.001% (vol / vol); (h) monobasic sodium phosphate monohydrate at a concentration of about 0.0278 g / L, and (i) Sodium phosphate dibasic anhydrous at a concentration of about 0.114 g / L.

[0032] In certain embodiments, the vector genome concentration (VGC) of the pharmaceutical composition is about 1 x 10 11 GC / mL, approximately 3×1011 GC / mL, approximately 6×10 11 GC / mL, approximately 1×10 12 G C / mL, approximately 3 × 10 12 GC / mL, approximately 6×10 12 GC / mL, approximately 1×10 13 GC / mL, approximately 2×10 13 GC / mL, approximately 3×10 13 GC / mL, approximately 4×10 13 GC / mL, approx. 5 x 10 13 GC / mL, approximately 6×10 13 GC / mL, approximately 7×10 13 GC / m L, about 8 x 10 13 GC / mL, approximately 9×10 13 GC / mL, or approximately 1 x 10 14 GC / mL, approximately 3×10 14 GC / mL, approximately 6×10 14 GC / mL, or approximately 1 x 10 15 GC / mL.

[0033] In certain embodiments, the pH of the pharmaceutical composition is in the range of about 6.0 to about 9.0. In embodiments, the pH of the pharmaceutical composition is about 7.4.

[0034] In certain embodiments, the rAAV in the pharmaceutical composition contains the same recombinant AAV as in the reference pharmaceutical composition. At least 2%, 5%, 7%, 10%, or 10% of freeze / thaw cycles compared to rAAV 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2x, 3x, 5x, 10x, 100x, or 1000x stable. In embodiments, the stability of the rAAV in a pharmaceutical composition is determined by: (a) rAAV infectivity, (b) rAAV aggregation levels, or (c) Levels of free DNA released by rAAV.

[0035] In certain embodiments, the pharmaceutical composition is a liquid composition. In certain embodiments, the pharmaceutical composition is a lyophilized composition or A reconstituted lyophilized composition.

[0036] In certain embodiments, the pharmaceutical composition is administered intracerebroventricularly (ICV), intracisternally (IC), intrathecally, Suitable for intracranial, intravenous, intravascular, intraarterial, intramuscular, intraocular, intramuscular, subcutaneous, or intradermal administration It has the following characteristics.

[0037] In certain embodiments, the coding sequence of (iii) of the rAAV in the pharmaceutical composition is The human CLN2 is an optimized human CLN2, which has at least one amino acid sequence similar to the native human coding sequence of SEQ ID NO:2. In certain embodiments, the rAAV in the pharmaceutical composition is at least 70% identical to (iii). The coding sequence is SEQ ID NO:3.

[0038] In certain embodiments, the rAAV capsid of the rAAV in the pharmaceutical composition is AAV9 or is its variant.

[0039] In certain embodiments, the promoter of the rAAV in the pharmaceutical composition is chicken β-actin In certain embodiments, the promoter of the rAAV in the pharmaceutical composition is a CBA promoter. The promoter contains the CBA promoter sequence and the cytomegalovirus enhancer element. It is a hybrid promoter.

[0040] In certain embodiments, the AAV 5'ITR and / or AA of the rAAV in the pharmaceutical composition The V 3'ITR is derived from AAV2.

[0041] In certain embodiments, the vector genome of the rAAV in the pharmaceutical composition further comprises polyA. In certain embodiments, the poly A is synthetic poly A or bovine growth hormone (BGH) GH), human growth hormone (hGH), SV40, rabbit β-globin (RGB), or is derived from modified RGB (mRGB).

[0042] In certain embodiments, the vector genome of the rAAV in the pharmaceutical composition further comprises an intron. In certain embodiments, the introns include CBA, human beta globin, IVS2, SV 40, bGH, alpha globulin, beta globulin, collagen, ovalbumin, or p5 3. It comes from

[0043] In certain embodiments, the vector genome of the rAAV in the pharmaceutical composition further comprises an enhancer. In certain embodiments, the enhancer includes a CMV enhancer, a RSV enhancer, or a Sir, APB enhancer, ABPS enhancer, αmic / bik enhancer, T TR enhancer, en34, and ApoE.

[0044] In certain embodiments, the rAAV vector genome in the pharmaceutical composition is about 3 kilograms in size. In certain embodiments, the base of the rAAV in the pharmaceutical composition is between about 5.5 kilobases. The ectodermal genome is approximately 4 kilobases in size.

[0045] In certain embodiments, suspension cell lines capable of producing rAAV are grown in suspension culture. The rAAV is produced in a pharmaceutical composition using a method comprising:

[0046] In another aspect, a method for treating a patient comprising administering to the patient a pharmaceutical composition provided herein. Provided herein are methods for treating CLN2 Batten disease in a patient. The pharmaceutical composition is administered in a therapeutically effective amount. In certain embodiments, the subject is a human.

[0047] In yet another aspect, provided herein is a kit comprising one or more containers and instructions for use. wherein one or more containers contain a pharmaceutical composition provided herein.

[0048] Also provided herein are methods for producing rAAV. In certain embodiments, the methods include: This involves growing a suspension cell line capable of producing AAV in suspension culture.

[0049] Other aspects and embodiments will be readily apparent based on the information provided herein. cormorant. Exemplary Embodiments 1. A method for treating CLN2 Batten disease in a subject, comprising administering to a subject in need thereof Administration of recombinant adeno-associated virus (rAAV) via the first and second routes wherein the first pathway and the second pathway are pathways to the central nervous system (CNS), the first pathway is a pathway to a brain region, and the second pathway is a pathway to a spinal cord region; and The recombinant adeno-associated virus (rAAV) contains the AAV capsid and the packaged a vector genome encoded therein, said vector genome comprising: (a) AAV 5′ inverted terminal repeat (ITR) sequence; (b) promoter; (c) the CLN2 coding sequence encoding human TPP1; and (d) The above therapeutic method, which comprises an AAV 3'ITR. 2. The method according to item 1, wherein the first route is intracerebroventricular (ICV) or intracisternal (IC). How to do it. 3. Any one of items 1 to 2, wherein the second route is intrathecal (IT-L) lumbar. The method described below. 4. The method further comprises administering the rAAV to the subject via a third route. and the third route includes intracerebroventricular (ICV), intracisternal (IC), lumbar intrathecal, intracranial, intravenous, an item selected from the group consisting of intravenous, intravascular, intraarterial, intramuscular, intraocular, subcutaneous, and intradermal The method according to any one of 1 to 3. 5. A method for treating CLN2 Batten disease in a subject, comprising administering to a subject in need thereof Administration of recombinant adeno-associated virus (rAAV) via the first and second routes wherein the first pathway is to the central nervous system (CNS) and the second pathway delivers the rAAV to the liver; and The recombinant adeno-associated virus (rAAV) contains the AAV capsid and the packaged a vector genome encoded therein, said vector genome comprising: (a) AAV 5′ inverted terminal repeat (ITR) sequence; (b) promoter; (c) the CLN2 coding sequence encoding human TPP1; and (d) The above therapeutic method, which comprises an AAV 3'ITR. 6. The first route is intrathecal (IT-L), intracerebroventricular (ICV), or intracisternal (ICV) The method according to item 5, wherein C). 7. The second route is selected from the group consisting of intravenous, intravascular, intraarterial, intramuscular, intraocular, subcutaneous, and intradermal. The method according to any one of items 5 to 6, wherein the compound is selected from the group consisting of: 8. The method of item 7, wherein the second route is intravenous. 9. The method comprises administering the rAAV via the second route while simultaneously administering the 9. The method according to any one of items 1 to 8, comprising administering the rAAV via one of the following routes: How to do it. 10. The method further comprises administering the rAAV via the first route prior to administering the rAAV via the second route. 9. The method according to any one of items 1 to 8, comprising administering the rAAV via the following route: method. 11. The method further comprises administering the rAAV via the second route followed by administering the first route. 9. The method according to any one of items 1 to 8, comprising administering the rAAV via the following route: method. 12. Administering the rAAV via the first route and the rAAV via the second route. The interval between administrations of rAAV is about 0.5 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, or 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 8 days, about 9 Days, about 10 days, about 11 days, about 12 days, about 13 days, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks About 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, About 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, or more 12. The method according to any one of Items 10 to 11. 13. The method further comprises measuring, in the spinal cord of the subject, a reference TPP in the spinal cord of a second subject. Compared to 1 activity, at least 2%, 3%, 5%, 6%, 7%, 8%, 9%, 10%, 12 %, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% and providing a high TPP1 activity in the spinal cord, wherein the reference TPP1 activity in the spinal cord is higher than the second counterpart. the first subject is not treated using the method, and the second subject is 13. The method according to any one of items 1 to 12, wherein the phenotype is the same as or different from the phenotype of the elephant. 14. The method comprises determining the level of TPP1 activity in a subject by at least 2%, 3%, or 4% of the reference liver TPP1 activity in a second subject. %, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25% 30%, 35%, 40%, 45%, or 50% higher hepatic TPP1 activity in said subject. and said reference hepatic TPP1 activity is greater than or equal to said second subject receiving treatment using said method. wherein the second subject is the same as or different from the first subject; 14. The method according to any one of items 1 to 13. 15. The method comprises determining whether the serum TPP1 activity of the subject is at least 2% relative to a reference serum TPP1 activity of the second subject; 3%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25 %, 30%, 35%, 40%, 45%, or 50% higher serum TPP1 activity in said subject. and said reference serum TPP1 activity is greater than or equal to said first subject's serum TPP1 activity after said second subject has been treated using said method. and the second subject is the same or different from the first subject. 15. The method according to any one of items 1 to 14, wherein 16. The method further comprises: obtaining, in the cortex of the subject, a reference micrograph in the cortex of a second subject; At least 2%, 3%, 5%, 6%, 7%, 8%, 9%, 10% of glial activity 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, or 5 0% lower microglial activity, and the reference microglial activity in the cortex is the second subject is not receiving treatment using the method, 16. The method according to any one of items 1 to 15, wherein the subject is the same as or different from the subject. How to do it. 17. The method further comprises measuring, in the brain of the subject, a reference TPP1 activity in the brain of a second subject. At least 2%, 3%, 5%, 6%, 7%, 8%, 9%, 10%, 12% compared to sex 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% higher and wherein said reference TPP1 activity in said brain is determined to be in a range of from 0.01 to 0.01 in said second subject. and the second subject is not receiving treatment using the method, and the second subject is 17. The method according to any one of items 1 to 16, wherein the 18. The method according to any one of items 1 to 17, wherein the rAAV is administered in a therapeutically effective amount. method. 19. The method of any one of items 1 to 18, wherein the subject is a human. 20. The coding sequence of (c) is at least 200kJ / 300kcal with respect to the native human coding sequence of SEQ ID NO:2. 19. A codon-optimized human CLN2 that is 70% identical to the human CLN2 of any one of items 1 to 19. The method described. 21. The method according to any one of items 1 to 20, wherein the coding sequence of (c) is SEQ ID NO: 3. The method described. 22. The method of any one of claims 1 to 10, wherein the rAAV capsid is AAV9 or a variant thereof. 21. The method of any one of claims 1 to 21. 23. The promoter is a chicken beta actin (CBA) promoter. 23. The method according to any one of 1 to 22. 24. The promoter comprises a CBA promoter sequence and a cytomegalovirus enhancer. 24. Any one of items 1 to 23, wherein the promoter is a hybrid promoter containing a sensor element. The method described below. 25. The AAV 5'ITR and / or AAV 3'ITR are derived from AAV2. The method according to any one of Items 1 to 24. 26. The vector genome according to any one of items 1 to 25, wherein the vector genome further comprises polyA. method. 27. The poly A is synthetic poly A or bovine growth hormone (bGH), human growth hormone (HGH), or Human growth hormone (hGH), SV40, rabbit β-globin (RGB), or modified R 27. The method according to item 26, wherein the chromatin is derived from GB (mRGB). 28. The vector genome according to any one of items 1 to 27, wherein the vector genome further comprises an intron. How to post. 29. The intron is selected from CBA, human β-globin, IVS2, SV40, bGH, derived from alpha globulin, beta globulin, collagen, ovalbumin, or p53; Item 29. The method according to item 28. 30. The method according to any one of items 1 to 29, wherein the vector genome further comprises an enhancer. The method described. 31. The enhancer is a CMV enhancer, an RSV enhancer, or an APB enhancer. enhancer, ABPS enhancer, αmic / bik enhancer, TTR enhancer Item 31. The method according to item 30, wherein the antibody is selected from the group consisting of en34, ApoE, and en34. 32. The size of the vector genome is about 3 kilobases to about 5.5 kilobases. 32. The method according to any one of Items 1 to 31. 33. Any of items 1 to 32, wherein the vector genome is about 4 kilobases in size. The method described above. 34. Propagating the suspension cell line capable of producing the rAAV in suspension culture. 34. The method according to any one of items 1 to 33, wherein the rAAV is produced using a method comprising: . 35. The method of item 34, wherein the suspension cell line is a HEK293 suspension cell line. 36. (a) recombinant adeno-associated virus (rAAV); (b) sodium chloride, (c) magnesium chloride, (d) potassium chloride, (e) dextrose, (f) Poloxamer 188, (g) monobasic sodium phosphate, and (h) A pharmaceutical composition comprising dibasic sodium phosphate, The recombinant adeno-associated virus (rAAV) contains the AAV capsid and the packaged and a vector genome encoded therein, said vector genome comprising: (i) an AAV 5' inverted end (ii) the ITR sequence; (iii) the promoter; and (iv) the CLN2 encoding human TPP1. a coding sequence; and (iv) an AAV 3' ITR. 37. The pharmaceutical composition according to item 36, further comprising calcium chloride. 38. The sodium chloride, the magnesium chloride, the potassium chloride, the dextran Trose, Poloxamer 188, Sodium Phosphate Monobasic, Sodium Phosphate Dibasic and the calcium chloride are present in the form of anhydrous, monohydrate, dihydrate, trihydrate, tetrahydrate, Hydrate, pentahydrate, hexahydrate, heptahydrate, octahydrate, nonahydrate, or decahydrate forms Item 38. The pharmaceutical composition according to Item 37, wherein 39. (a) the rAAV; (b) sodium chloride at a concentration of about 8.77 g / L; (c) magnesium chloride hexahydrate at a concentration of about 0.244 g / L; (d) potassium chloride at a concentration of about 0.224 g / L; (e) calcium chloride dihydrate at a concentration of about 0.206 g / L; (f) anhydrous dextrose at a concentration of about 0.793 g / L; (g) poloxamer 188 at a concentration of about 0.001% (vol / vol); (h) monobasic sodium phosphate monohydrate at a concentration of about 0.0278 g / L, and (i) Items 36 to 38, containing dibasic sodium phosphate anhydrous at a concentration of about 0.114 g / L The pharmaceutical composition according to any one of the preceding claims. 40. The vector genome concentration (VGC) of the pharmaceutical composition is about 1 x 10 11 GC / m L, about 3 x 10 11 GC / mL, approximately 6×10 11 GC / mL, approximately 1×10 12 GC / mL , about 3×10 12 GC / mL, approximately 6×10 12 GC / mL, approximately 1×10 13 GC / mL, Approximately 2×10 13 GC / mL, approximately 3×10 13 GC / mL, approximately 4×10 13 GC / mL, approx. 5×10 13 GC / mL, approximately 6×10 13 GC / mL, approximately 7×10 13 GC / mL, approx. 8 x10 13 GC / mL, approximately 9×10 13 GC / mL, or approximately 1 x 10 14 GC / mL, Approximately 3×10 14 GC / mL, approximately 6×10 14 GC / mL, or approximately 1 x 10 15 GC / m 40. The pharmaceutical composition according to any one of items 36 to 39, wherein L. 41. The pharmaceutical composition according to any one of items 36 to 40, wherein the pH of the pharmaceutical composition is in the range of about 6.0 to about 9.0. The pharmaceutical composition according to any one of the preceding claims. 42. The pharmaceutical composition according to item 41, wherein the pH of the pharmaceutical composition is about 7.4. 43. The rAAV in the pharmaceutical composition is the same recombinant rAAV in a reference pharmaceutical composition. at least 2%, 5%, 7%, 10%, and 12% for freeze / thaw cycles compared to , 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100 %, 2x, 3x, 5x, 10x, 100x, or 1000x stable, items 36-4 3. The pharmaceutical composition according to any one of claims 2. 44. The stability of the rAAV is (a) rAAV infectivity, (b) rAAV aggregation levels, or (c) As determined by the level of free DNA released by rAAV, see item 43. The pharmaceutical composition described. 45. The pharmaceutical composition according to any one of items 36 to 44, wherein the pharmaceutical composition is a liquid composition. Pharmaceutical compositions. 46. ​​The pharmaceutical composition according to any one of items 36 to 44, wherein the pharmaceutical composition is a frozen composition. Pharmaceutical compositions. 47. The pharmaceutical composition is a lyophilized composition or a reconstituted lyophilized composition. 45. The pharmaceutical composition according to any one of items 36 to 44. 48. The pharmaceutical composition is administered intracerebroventricularly (ICV), intracisternally (IC), intrathecally, intracranially, Suitable for intravenous, intravascular, intraarterial, intramuscular, intraocular, intramuscular, subcutaneous, or intradermal administration 48. The pharmaceutical composition according to any one of items 36 to 47, comprising: 49. The coding sequence of (iii) is at least 100% identical to the native human coding sequence of SEQ ID NO:2. any of items 36 to 48, which is a codon-optimized human CLN2 that is at least 70% identical to The pharmaceutical composition described in claim 1. 50. Any of items 36 to 49, wherein the coding sequence of (iii) is SEQ ID NO: 3. The pharmaceutical composition described in claim 1. 51. Item 36, wherein the rAAV capsid is AAV9 or a variant thereof. A pharmaceutical composition described in any one of claims 1 to 50. 52. The promoter is a chicken beta actin (CBA) promoter. 52. The pharmaceutical composition according to any one of claims 36 to 51. 53. The promoter comprises a CBA promoter sequence and a cytomegalovirus enhancer. 53. The method according to any one of items 36 to 52, wherein the promoter is a hybrid promoter containing a sensor element. The pharmaceutical composition according to item 1. 54. The AAV 5'ITR and / or AAV 3'ITR are derived from AAV2. The pharmaceutical composition according to any one of Items 36 to 53, 55. Any one of items 36 to 54, wherein the vector genome further comprises polyA. The pharmaceutical composition described. 56. The poly A is synthetic poly A or bovine growth hormone (bGH), human growth hormone (HGH), or Human growth hormone (hGH), SV40, rabbit β-globin (RGB), or modified R 56. The pharmaceutical composition according to any one of items 36 to 55, which is derived from GB (mRGB). 57. Any one of items 36 to 56, wherein the vector genome further comprises an intron. The pharmaceutical composition according to item 1. 58. The intron is selected from CBA, human β-globin, IVS2, SV40, bGH, derived from alpha globulin, beta globulin, collagen, ovalbumin, or p53; 58. The pharmaceutical composition according to any one of items 36 to 57. 59. Any of items 36 to 58, wherein the vector genome further comprises an enhancer. The pharmaceutical composition described in claim 1. 60. The enhancer is a CMV enhancer, an RSV enhancer, or an APB enhancer. enhancer, ABPS enhancer, αmic / bik enhancer, TTR enhancer 59. The pharmaceutical composition according to any one of items 36 to 59, wherein the antibody is selected from the group consisting of α- and β-actin, α-actin, β ... 61. The size of the vector genome is about 3 kilobases to about 5.5 kilobases. 61. The pharmaceutical composition according to any one of Items 36 to 60. 62. Any of items 36 to 61, wherein the size of the vector genome is about 4 kilobases. The pharmaceutical composition according to any one of claims 1 to 4. 63. Propagating the suspension cell line capable of producing the rAAV in suspension culture. 63. The method of any one of items 36 to 62, wherein the rAAV is produced using a method comprising: A pharmaceutical composition comprising: 64. A method for treating a disease comprising administering the pharmaceutical composition according to any one of items 36 to 63 to a subject. a method for treating CLN2 Batten disease in said subject. 65. The method according to item 64, wherein the pharmaceutical composition is administered in a therapeutically effective amount. 66. The method of any one of items 64 to 65, wherein the subject is a human. 67. A kit comprising one or more containers and instructions for use, wherein the one or more containers 64. The kit comprising the pharmaceutical composition according to any one of Items 36 to 63. [Brief explanation of the drawings]

[0050] [Figure 1A]

[0039] Figure 1 is a schematic diagram of the AAV.CB7.CI.hTPP1co.RBG vector genome. ITR represents the AAV2 inverted terminal repeat sequence. CB7 represents the chicken β-actin promoter with the cytomegalovirus enhancer. RBG polyA represents the rabbit β-globin polyadenylation signal.

[0051] [Figure 1B] 1 shows a map of the production plasmid for the AAV.hTPP1co vector.

[0052] [Figure 1C] A map of the AAV cis-plasmid construct is shown. 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 β-globin polyadenylation signal; Kan-r: kanamycin resistance gene.

[0053] [Figure 1D] A map of the AAV trans-packaging plasmid construct is shown.

[0054] [Figure 1E] 1 shows a map of the adenovirus helper plasmid.

[0055] [Figure 2] 1 shows increased survival in TPP1m1J knockout mice treated with AAV9.CB7.hCLN2.

[0056] [Figure 3] Figure 1 shows increased TPP1 activity in the brains of AAV9.CB7.hCLN2-treated TPP1m1J knockout mice. *p<0.05; **p<0.01 compared to untreated TPP1m1J knockout mice using Wilcoxon test. Individual values ​​are shown along with the mean and SEM.

[0057] [Figure 4] Figure 1 shows increased TPP1 activity in the spinal cord of AAV9.CB7.hCLN2-treated TPP1m1J knockout mice. **p<0.01 compared to untreated TPP1m1J knockout mice using Wilcoxon test. Individual values ​​are shown along with the mean and SEM.

[0058] [Figure 5] Figure 1 shows that astrocytosis was reduced in TPP1m1J knockout mice treated with AAV9.CB7.hCLN2. *p<0.05; **p<0.01 compared to untreated TPP1m1J knockout mice using one-way ANOVA. Individual values ​​are shown along with the mean and SEM.

[0059] [Figure 6]Figure 1 shows that microglial activation was reduced in TPP1m1J knockout mice treated with AAV9.CB7.hCLN2. *p<0.05; **p<0.01 compared to untreated TPP1m1J knockout mice using one-way ANOVA. Individual values ​​are shown along with the mean and SEM.

[0060] [Figure 7] Figure 1 shows increased hepatic TPP1 activity in AAV9.CB7.hCLN2-treated TPP1m1J knockout mice. *p<0.05; **p<0.01 compared to untreated TPP1m1J knockout mice using Wilcoxon test. Individual values ​​are shown along with the mean and SEM.

[0061] [Figure 8] Serum TPP1 activity was increased in AAV9.CB7.hCLN2-treated TPP1m1J knockout mice. *p<0.05; **p<0.01 compared to untreated TPP1m1J knockout mice using Wilcoxon test. Individual values ​​are shown along with the mean and SEM.

[0062] [Figure 9] Figure 1 shows that AAV9.CB7.hCLN2 therapy increased survival in knockout animals after treatment with HD at 1 month of age. All surviving animals were necropsied 26 weeks after ICV, and no differences were observed between controls and LD-treated knockouts. WT males (filled circles), WT females (gray squares), and HD KO males (filled crosses) are overlaid on the plots because no deaths were observed in these three groups. WT: wild-type; KO: TPP1m1J knockout; M: male mice; F: female mice; LD: low dose (3 × 10 GC / animal); HD: high dose (3 × 10 GC / animal).

[0063] [Figure 10A]Normalization of astrocytosis is demonstrated. A. Brainstem. Results are the average number of astrocytes per 20x field. "KO PBS-treated" (no ICV) are 3-month-old (pre-symptomatic) animals obtained from a natural history study (W2553A). P values ​​by unpaired Mann-Whitney test. WT: wild-type; KO: TPP1m1J knockout; and HD: high dose (3 x 10 GC / animal). [Figure 10B] Normalization of astrocytosis is shown. B. Hippocampus. Results are the average number of astrocytes per 20x field. "KO PBS-treated" (no ICV) are 3-month-old (presymptomatic) animals obtained from a natural history study (W2553A). P values ​​by unpaired Mann-Whitney test. WT: wild-type; KO: TPP1m1J knockout; and HD: high dose (3 x 10 GC / animal). [Figure 10C] Normalization of astrocytosis is shown. C. Cortex. Results are the average number of astrocytes per 20x field. "KO PBS-treated" (no ICV) are 3-month-old (presymptomatic) animals obtained from a natural history study (W2553A). P values ​​by unpaired Mann-Whitney test. WT: wild-type; KO: TPP1m1J knockout; and HD: high dose (3 x 10 GC / animal).

[0064] [Figure 11] Survival data are shown. All vehicle-treated TPP1m1J knockout mice were confirmed dead or humanely euthanized before 19 weeks of age, while 67% of AAV9.CB7.hCLN2-treated females (3×10 GC / animal) and 57% of AAV9.CB7.hCLN2-treated males (3×10 GC / animal) survived to the planned 23-week endpoint. KO3e11 M: AAV9.CB7.hCLN2-treated male knockout (3×10 GC / animal); KO3e11 F: AAV9.CB7.hCLN2-treated female knockout (3×10 GC / animal); KO M: vehicle-treated male knockout; KO F: vehicle-treated female knockout; WT M: wild-type male; WT F: wild-type female.

[0065] [Figure 12] Figure 1 shows that AAV9.CB7.hCLN2 increased survival in TPP1m1J knockout mice. Groups of TPP1m1J knockout mice were administered doses of 0, 1.25x10, 5x10, 2x10, or 8.5x10 GC / animal. An additional group of WT mice was untreated (study ongoing).

[0066] [Figure 13] Figure 1 shows that AAV9.CB7.hCLN2 increased TPP1 activity in the brains of TPP1m1J knockout mice. Groups of TPP1m1J knockout mice were administered doses of 0, 1.25 x 10, 5 x 10, 2 x 10, or 8.5 x 10 GC / animal. An additional group of WT mice was untreated. At 13 weeks of age (week 9), animals were euthanized, and TPP1 activity was analyzed in the right hemisphere of the brain for A) males and B) females. *p ≤ 0.05; **p ≤ 0.01. P values ​​were obtained using a two-tailed Wilcoxon exact rank-sum test, comparing each treatment group to an independent control group under the null hypothesis of no difference between the two groups.

[0067] [Figure 14] Figure 1 shows that AAV9.CB7.hCLN2 increased TPP1 activity in the spinal cord of TPP1m1J knockout mice. Groups of TPP1m1J knockout mice were administered doses of 0, 1.25 x 10, 5 x 10, 2 x 10, or 8.5 x 10 GC / animal. An additional group of WT mice was untreated. At 13 weeks of age (week 9), animals were euthanized and the spinal cord (thoracic) was analyzed for TPP1 activity. Black represents males, and gray represents females. Due to the limited number of samples analyzed, data from males and females were combined. *p ≤ 0.05; **p ≤ 0.01. P values ​​were obtained using a two-tailed Wilcoxon exact rank-sum test, comparing each treatment group to an independent control group under the null hypothesis of no difference between the two groups.

[0068] [Figure 15]Figure 1 shows that AAV9.CB7.hCLN2 increased TPP1 activity in the livers of TPP1m1J knockout mice. Groups of TPP1m1J knockout mice were administered doses of 0, 1.25 x 10, 5 x 10, 2 x 10, or 8.5 x 10 GC / animal. An additional group of WT mice was untreated. At 13 weeks of age (week 9), animals were euthanized, and liver TPP1 activity was analyzed for A) males and B) females. *p ≤ 0.05; **p ≤ 0.01. P values ​​were obtained using a two-tailed Wilcoxon exact rank-sum test, comparing each treatment group to an independent control group under the null hypothesis of no difference between the two groups.

[0069] [Figure 16] Figure 1 shows that AAV9.CB7.hCLN2 increased TPP1 activity in the serum of TPP1m1J knockout mice. Groups of TPP1m1J knockout mice were administered doses of 0, 1.25 x 10, 5 x 10, 2 x 10, or 8.5 x 10 GC / animal. An additional group of WT mice was untreated. At 13 weeks of age (week 9), animals were euthanized and blood was collected for serum TPP1 activity for A) males and B) females. *p ≤ 0.05; **p ≤ 0.01. P values ​​were obtained using a two-tailed Wilcoxon exact rank-sum test, comparing each treatment group to an independent control group under the null hypothesis of no difference between the two groups.

[0070] [Figure 17] Figure 1 shows that AAV9.CB7.hCLN2 reduced astrocytosis in TPP1m1J knockout mice. Groups of TPP1m1J knockout mice were administered doses of 0 (group 2), 1.25 x 10 (group 3), 5 x 10 (group 4), 2 x 10 (group 5), or 8.5 x 10 GC / animal (group 6). An additional group of WT mice was untreated (group 1). Brain sections were stained for GFAP to measure relative levels of immunofluorescence in astrocytes in A) the somatosensory barrel cortex (S1BF) and B) the thalamus (ventral posterolateral nucleus [VPL] and ventral posteromedial nucleus [VPM]). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

[0071] [Figure 18] Figure 1 shows that AAV9.CB7.hCLN2 reduced microglial activation in TPP1m1J knockout mice. Groups of TPP1m1J knockout mice were administered doses of 0 (group 2), 1.25 x 10 (group 3), 5 x 10 (group 4), 2 x 10 (group 5), or 8.5 x 10 GC / animal (group 6). An additional group of WT mice was untreated (group 1). Brain sections were stained with CD68 to measure relative levels of immunofluorescence in microglia in A) the somatosensory barrel cortex (S1BF) and B) the thalamus (ventral posterolateral nucleus [VPL] and ventral posteromedial nucleus [VPM]).

[0072] [Figure 19] Figure 1 shows TPP1 activity in serum from C57Bl / 6 mice. Groups of C57Bl / 6 mice were administered doses of 0, 1.25x10, 5x10, 2x10, or 8.5x10 GC / animal. Four or 13 weeks after administration, animals were euthanized and blood was collected for serum TPP1 activity for A) males and B) females. *p≤0.05; **p≤0.01 vs. time-matched control group. P values ​​were obtained using a two-tailed Wilcoxon exact rank-sum test, comparing each treatment group to an independent control group under the null hypothesis of no difference between the two groups.

[0073] [Figure 20] Figure 1 shows TPP1 activity in the brains of C57Bl / 6 mice. Groups of C57Bl / 6 mice were administered doses of 0, 1.25 x 10, 5 x 10, 2 x 10, or 8.5 x 10 GC / animal. Animals were euthanized 4 or 13 weeks after administration, and brains were collected for TPP1 activity for A) males and B) females. *p ≤ 0.05; **p ≤ 0.01 vs. time-matched control group. P values ​​were obtained using a two-tailed Wilcoxon exact rank-sum test, comparing each treatment group to an independent control group under the null hypothesis of no difference between the two groups.

[0074] [Figure 21]Figure 1 shows TPP1 activity in the livers of C57Bl / 6 mice. Groups of C57Bl / 6 mice were administered doses of 0, 1.25 x 10, 5 x 10, 2 x 10, or 8.5 x 10 GC / animal. Four or 13 weeks after administration, animals were euthanized, and livers were harvested for TPP1 activity for A) males and B) females. *p ≤ 0.05; **p ≤ 0.01 vs. time-matched control group. P values ​​were obtained using a two-tailed Wilcoxon exact rank-sum test, comparing each treatment group to an independent control group under the null hypothesis of no difference between the two groups.

[0075] [Figure 22] Differences from baseline in serum TPP1 activity and concentration are shown. Groups of cynomolgus monkeys (n=3 / dose) were administered AAV9.CB7.hCLN2 via injection into the cisterna magna (CM) at doses of 0, 3.4×10, 3.2×10, or 2.9×10 genome copies (GC) / animal (1 mL dose). Blood samples were collected pre-dose (day -1 or day 1) and on days 4, 8, 11, 15, 18, 22, 25, and 29 for analysis of (A) TPP1 activity and (B) TPP1 concentration. Mean differences from baseline are shown along with the standard error of the mean.

[0076] [Figure 23] Differences from baseline in TPP1 activity and concentration in CSF are shown. Groups of cynomolgus monkeys (n=3 / dose) were administered AAV9.CB7.HCLN2 via injection into the cisterna magna (CM) at doses of 0, 3.4×10 11 , 3.2×10 12 , or 2.9×10 13 genome copies (GC) / animal (1 mL dose). CSF samples were collected pre-dose (day -1 or day 1) and on days 4, 8, 11, 15, 18, 22, 25, and 29 for analysis of (A) TPP1 activity and (B) TPP1 concentration. Mean differences from baseline are shown along with the standard error of the mean.

[0077] [Figure 24A]TPP1 concentrations in the brain region frontal cortex are shown. Groups of cynomolgus monkeys (n=3 / dose) were administered AAV9.CB7.HCLN2 via injection into the cisterna magna (CM) at doses of 0, 3.4 x 10, 3.2 x 10, or 2.9 x 10 genome copies (GC) / animal (1 mL dose). At necropsy on day 29, two tissue punches were taken from the deep (>3 mm; D) or superficial (<3 mm deep; S) regions of the frontal cortex. Individual values ​​are shown along with the mean and standard deviation. [Figure 24B] TPP1 concentrations in the striatum, a brain region, are shown. Groups of cynomolgus monkeys (n=3 / dose) were administered AAV9.CB7.HCLN2 via injection into the cisterna magna (CM) at doses of 0, 3.4 x 10, 3.2 x 10, or 2.9 x 10 genome copies (GC) / animal (1 mL dose). Two tissue punches were taken from the deep (>3 mm; D) or superficial (<3 mm deep; S) regions of the striatum at necropsy on day 29. Individual values ​​are shown along with the mean and standard deviation. [Figure 24C] TPP1 concentrations in the brain region thalamus are shown. Groups of cynomolgus monkeys (n=3 / dose) were administered AAV9.CB7.HCLN2 via injection into the cisterna magna (CM) at doses of 0, 3.4 x 10, 3.2 x 10, or 2.9 x 10 genome copies (GC) / animal (1 mL dose). Two tissue punches were taken from the deep (>3 mm; D) or superficial (<3 mm deep; S) regions of the thalamus at necropsy on day 29. Individual values ​​are shown along with the mean and standard deviation. [Figure 24D] TPP1 concentrations in the brain region midbrain are shown. Groups of cynomolgus monkeys (n=3 / dose) were administered AAV9.CB7.HCLN2 via injection into the cisterna magna (CM) at doses of 0, 3.4 x 10, 3.2 x 10, or 2.9 x 10 genome copies (GC) / animal (1 mL dose). Two tissue punches were taken from the deep (>3 mm; D) or superficial (<3 mm deep; S) regions of the midbrain at necropsy on day 29. Individual values ​​are shown along with the mean and standard deviation.

[0078] [Figure 25]TPP1 concentrations are shown in the brain regions (A) occipital cortex, (B) medulla oblongata, and (C) cerebellum. Groups of cynomolgus monkeys (n = 3 / dose) were administered AAV9.CB7.HCLN2 via injection into the cisterna magna (CM) at doses of 0, 3.4 x 10, 3.2 x 10, or 2.9 x 10 genome copies (GC) / animal (1 mL dose). At necropsy on day 29, two tissue punches were taken from the deep (>3 mm; D) or superficial (<3 mm depth; S) regions of (A) occipital cortex, (B) medulla oblongata, and (C) cerebellum. Individual values ​​are shown along with the mean and standard deviation.

[0079] [Figure 26] TPP1 concentrations in the spinal cord are shown. Groups of cynomolgus monkeys (n = 3 / dose) were administered AAV9.CB7.HCLN2 via injection into the cisterna magna (CM) at doses of 0, 3.4 x 10, 3.2 x 10, or 2.9 x 10 genome copies (GC) / animal (1 mL dose). At necropsy on day 29, two tissue punches were taken from either the cervical, thoracic, or lumbar region of the spinal cord for (A) TPP1 activity and (B) TPP1 concentration. Individual values ​​are shown along with the mean and standard deviation.

[0080] [Figure 27] Shown is the temperature profile measured at two different fill volumes of a Nalgene HDPE BDS bottle.

[0081] [Figure 28] Shown are temperature profiles recorded for 0.6 mL fills of 2 mL cryovials cycled between -80°C and room temperature or -20°C.

[0082] [Figure 29] Shows fast freeze / fast thaw (FF / FT) temperature characteristics.

[0083] [Figure 30] Fast freeze / fast thaw (FF / FT) temperature profile (left axis) and rate of change of shelf and probe temperatures (right axis) are shown.

[0084] [Figure 31] Shows fast freeze / slow thaw (FF / ST) temperature characteristics.

[0085] [Figure 32] Shows the temperature characteristics of slow freezing / fast thawing (SF / FT).

[0086] [Figure 33] Slow freeze / slow thaw (SF / ST) temperature characteristics are shown.

[0087] [Figure 34] The slow freeze / slow thaw (SF / ST) temperature profile (left axis) and the rate of change of shelf and probe temperatures (right axis) are shown.

[0088] [Figure 35] A magnified view of the SEC results characterization of AAV9.CB7.HCLN2 in intrathecal buffer is shown.

[0089] [Figure 36] 1 shows DLS diameter results for AAV9.CB7.HCLN2 freeze-thaw samples.

[0090] [Figure 37] FIG. 10 shows a low-temperature DSC thermogram of AAV9.CB7.HCLN2 modified Elliot's B formulation buffer.

[0091] [Figure 38] Figure 1 shows a flow diagram of the manufacturing of bulk drug substance (upstream).

[0092] [Figure 39] 1 is a flow diagram of the manufacturing of bulk drug substance (downstream). DETAILED DESCRIPTION OF THE INVENTION

[0093] Provided herein are methods and compositions for treating Batten disease. Suitable compositions include recombinant adeno-associated viruses (rAAVs), rAAVs containing AAV capsids, and and a vector genome packaged therein, the vector genome comprising: (a) AAV 5' inverted repeat (ITR) sequence; (b) promoter; (c) human TPP1 (d) the CLN2 coding sequence encoding the AAV 3' ITR (see Section I) The rAAV and related pharmaceutical compositions provided herein can be used to treat Batten disease. Methods for the preparation of medicaments for use in ... and administering the rAAV described herein to a subject in need thereof by multiple routes. (See Section II.) Also provided are methods for producing the rAAV described herein using suspension cell culture (I See Section II).

[0094] I. Recombinant Adeno-Associated Virus (rAAV) In certain embodiments, the AAV9.CB7.hCLN2 provided herein is As described in the embodiments below, the methods and compositions described herein can be used to treat NCL. and administering human tripeptidyl peptidase 1 (TPP1) protein to a subject in need thereof. In one embodiment, the present invention includes compositions and methods for delivering a CLN2 nucleic acid sequence encoding: Such compositions include codon-optimized versions of the CLN2 coding sequence, as shown in SEQ ID NO:3. It increases the efficacy of the product and therefore allows for the use of lower doses of reagents, thus improving safety. It is desirable to increase the activity of the naturally occurring CLN2 polypeptide as shown in SEQ ID NO:2. Compositions containing the code sequences are also included.

[0095] The TPP1 gene, also known as CLN2, has tripeptidyl peptidase I activity. It encodes tripeptidyl peptidase 1, a lysosomal serine protease that activates the lysosomal ATP-dependent ATP synthesis. It generates tripeptides from degradation products produced by lysosomal proteinases. It acts as a nonspecific lysosomal peptidase that requires substrates with unsubstituted N-termini. As used herein, the terms "TPP1," "CL "Tripeptidyl peptidase 1", "Tripeptidyl peptidase 2", and "Tripeptidyl peptidase 1" are used interchangeably when referring to the coding sequence. The naturally occurring nucleic acid sequence encoding human tripeptidyl peptidase 1 is available from NCB The sequence is reported in reference sequence NM_000391.3 and is reproduced herein in SEQ ID NO: 2. The two isoforms of human tripeptidyl peptidase 1 are UniProtK Reported under B / Swiss-Prot accession numbers O14773-1 and O14773-2 (reproduced herein as SEQ ID NOs: 1 and 4). Mutations are associated with late-infantile NCL (LINCL) disease.

[0096] In certain embodiments, the AAV.hTPP1co vector is prepared using the method described in WO2018209205 In certain embodiments, the human (h)TPP1 enzyme may be designed as described in A1. Coding-optimized cDNA was custom designed and synthesized for optimal codon usage. In a specific embodiment, the hTPP1co gene, reproduced as SEQ ID NO: 3, may then be used. The cDNA may be placed into a transgene expression cassette, which may be a cytomegalovirus (CMV) immediate early enhancer (C4) and chicken β-actin promoter The transcription from this promoter was driven by the CB7 promoter. , which is enhanced by the presence of the chicken β-actin intron (CI) (Figures 1A and 1B). In certain embodiments, the polyA signal of the expression cassette is derived from rabbit beta globin (RBG). It is poly A.

[0097] In a specific embodiment, the 6841 bp production plasmid of the AAV.hTPP1co vector (AAV.CB7.CI.hTPP1co.RBG) was synthesized using the hTPP1c The expression cassette is flanked by AAV2-derived ITRs and an amplicon as a selectable marker. In certain embodiments, the vector may be constructed with silencing resistance (Figure 1B). A similar AAV.hTPP1co production plasmid may also be constructed having the specific In embodiments, the vectors derived from both plasmids comprise the hTPP1c vector described herein. It is a single-stranded DNA genome containing an expression cassette and flanking ITRs derived from AAV2. It is also possible.

[0098] In certain embodiments, the AAV.hTPP1co vector is transfected using triple transfection. and containing 0.001% Pluronic F68 (PF68). The compound may be formulated in a vehicle consisting of phosphate buffered saline (PBS), for example, izukami, Hiroaki, et al., A Protocol for AA V vector production and purification,Dis s.Division of Genetic Therapeutics,Cen See Term for Molecular Medicine, 1998. In this embodiment, the genome titer of the produced vector is determined by droplet digital PCR ( It may also be measured via ddPCR. See, for example, M. Lock et al., Hu Ge ne Therapy Methods,Hum Gene Ther Methods .2014 Apr;25(2):115-25. doi: 10.1089 / hgt b.2013.131. Epub 2014 Feb 14.

[0099] Described herein is an exemplary AAV.hTPP1co vector, which is It may also be referred to as AAV9.CB7.hCLN2 in the specification. These terms have the same meaning. Additionally, in one embodiment, the AAV9.CB7.hCLN2 vector is Where applicable, alternative embodiments utilizing components as described herein are contemplated.

[0100] In certain embodiments of the invention, the subject has neuronal ceroid lipofuscinosis (NCL). , which the components, compositions, and methods of the present invention are designed to treat. As used herein, the term "subject" refers to a human, veterinary animal, or domestic animal, means mammals, including breeding animals or pets, and animals commonly used in clinical research. In embodiments, the subject of these methods and compositions is a human. Examples include, but are not limited to, mice, rats, dogs, cats, pigs, cows, sheep, and non-human primates. As used herein, the term "subject" is used interchangeably with "patient." It is used.

[0101] Neuronal ceroid lipofuscinosis (NCL) is a condition characterized by progressive intellectual and motor deterioration, It is a group of inherited neurodegenerative lysosomal storage disorders characterized by severe visual impairment, severe cognitive impairment, and early death. Loss of function is characteristic of most forms. Clinical phenotypes have traditionally been divided into infantile, late infantile-onset, and early-onset forms. Childhood, adult, and northern epilepsy (also known as progressive epilepsy with mental retardation [EPMR]) It has been characterized according to the age of onset and the order of appearance of the clinical features of the idiopathic encephalopathy (known as idiopathic encephalopathy). However, genetic and allelic heterogeneity exists; both the causative gene and the age of onset vary. New nomenclature and classification systems have been proposed and developed to take into account the For example, classic late-infantile CLN2 disease. First symptoms usually appear between the ages of 2 and 4 years. manifests, usually beginning with epilepsy, followed by developmental regression, myoclonic ataxia Visual impairment usually appears between the ages of 4 and 6 and progresses rapidly to impaired perception of light and dark. Life expectancy ranges from age 6 to early teens. The term "Batten disease" is used to refer to CLN2 disease, which is the same as "NCL." It is used in this sense.

[0102] In one aspect, a codon-optimized modified nucleic acid sequence encoding human (h)TPP1 is In certain embodiments, modified human (h)TPP1 cDNA is provided herein. (as SEQ ID NO:3), which sequence is shown to be The codon-optimized TPP1 coding sequence was designed to maximize translation. , less than about 80% identity to the full-length native TPP1 coding sequence (SEQ ID NO: 2), preferably or about 75% or less identity. In one embodiment, the codon-optimized TPP1 coding sequence The sequence has approximately 74% identity to the native TPP1 coding sequence of SEQ ID NO:2. In one embodiment, the codon-optimized TPP1 coding sequence is used for delivery via AAV (e.g., rAAV). In one embodiment, the codon-optimized TPP1 is characterized by a higher translation rate after transcription than that of native TPP1. The TPP1 coding sequence is approximately 99%, 98%, and 100% identical to the full-length native TPP1 coding sequence of SEQ ID NO:2. %, 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 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 has about 99%, 98%, or 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%, Share 67%, 66%, 65%, 64%, 63%, 62%, 61% or more identity In one embodiment, the codon-optimized nucleic acid sequence is SEQ ID NO: 3. In some embodiments, the nucleic acid sequence is codon-optimized for expression in humans. Select a suitable TPP1 coding sequence.

[0103] The terms "percent identity," "sequence identity," "percent sequence identity," and "sequence identity" in reference to nucleic acid sequences are used interchangeably. "Identical" or "~% identical" means the number of matches in two sequences when aligned to match. The length of sequence identity comparison can be the entire length of the genome, The full-length coding sequence or a fragment of at least about 500-5000 nucleotides. However, it is preferred that the sequence be, for example, at least about 9 nucleotides, usually at least About 20-24 nucleotides, at least about 28-32 nucleotides, at least about 36 Identity between smaller fragments of nucleotides or more may also be desired.

[0104] Protein, polypeptide, about 32 amino acids, about 330 amino acids, or peptides thereof The amino acid sequence or the corresponding nucleic acid sequence coding sequence of the full-length fragment of the The proportion of amino acids that can be readily determined is preferably at least about 8 amino acids in length. The "identity" between two different sequences is generally defined as a sequence of up to about 700 amino acids. When referring to "identity," "homology," or "similarity," "Similarity" is determined by reference to "aligned" sequences. "Alignment" refers to multiple nucleic acid sequences or protein (amino acid) sequences, see It often involves correction of deleted or additional bases or amino acids compared to the sequence.

[0105] Identity can be determined by preparing an alignment of the sequences and by methods known in the art. or by using various commercially available algorithms and / or computer programs. and can be determined by [e.g., BLAST, ExPASy; Clustal O;FASTA; e.g., Needleman-Wunsch algorithm, Smith -using the Waterman algorithm]. The alignment was performed using publicly available or using any of a variety of commercially available multiple sequence alignment programs. For example, "Clustal Omega," "Clustal X," "MAP," and "P IMA, MSA, BLOCKMAKER, MEME, and Match- Use a sequence alignment program such as the "Box" program on the amino acid sequence. Generally, you can use the default settings for both of these programs. However, those skilled in the art can change these settings as needed. At least the same level as that provided by the referenced algorithms and programs another algorithm or computer program that provides identity or alignment of sequences For example, JD Thomson et al., Nucl .Acids.Res.,“A comprehensive comparison of multiple sequence alignments”,27(13): 2682-2690 (1999).

[0106] Multiple sequence alignment programs are also available for nucleic acid sequences. Examples of useful programs include "Clustal Omega", "Clustal W", "CAP Sequence Assembly", "BLAST", "MAP", and "MEME" and other programs are accessed from web servers on the Internet. Other sources of such programs are known to those skilled in the art. The Vector NTI utility is also used. Any technique that can be used to measure nucleotide sequence identity, including algorithms There are also many algorithms known in the art. 6.1 program, Fasta™, to compare polynucleotide sequences. FASTA™ finds the best overlap between the query sequence and the search sequence. Provides alignment of overlapping regions and percent sequence identity. Percent sequence identity was calculated using GCG Version 6.1, which is incorporated herein by reference. As provided, Fasta™ is used with its default parameters (word size 6, and the NOPAM factor for the scoring matrix) can be used to determine Cut.

[0107] Codon-optimized coding regions can be designed by a variety of different methods. Optimizations were performed using methods available online (e.g., GeneArt) and published methods. or companies that provide codon optimization services, such as DNA2.0 (Menlo Park, rk, CA). One codon optimization method may be performed using, for example, No. WO2015 / 012924, which is incorporated herein by reference. See, for example, U.S. Patent Publication No. 2014 / 0032186 and U.S. Patent Publication No. See also Patent Application No. 2006 / 0136184. Preferably, the open reading of the product The entire length of the ORF is modified. However, in some embodiments, the ORF By using one of these methods, it is possible to modify only a fragment of any given Applying frequency to a polypeptide sequence, a codon-optimized coding region encoding the polypeptide is generated. Nucleic acid fragments of the range can be generated.

[0108] To perform the actual modifications to the codons or to codons designed as described herein Several options are available for synthesizing don-optimized code regions. Such modifications or synthesis can be carried out using standard and routine molecular biological manipulations well known to those skilled in the art. In one approach, the fragments are made up of fragments each 80-90 nucleotides in length. A series of complementary oligonucleotide pairs spanning the length of the desired sequence is prepared using standard These oligonucleotide pairs are synthesized by the method described above. When annealed, the cohesive ends are It is synthesized to form a double-stranded fragment of 80-90 base pairs including the ends, e.g., 3, 4, 5 , 6, 7, 8, 9, 10, or more bases in the other oligonucleotide of the pair. Each oligonucleotide in the pair is synthesized so that it extends beyond the complementary region. The single-stranded end of each oligonucleotide pair is connected to the single-stranded end of another oligonucleotide pair. The oligonucleotide pair is annealed and then Approximately 5-6 of these double-stranded fragments are annealed together via the single-stranded cohesive ends. They are then ligated together and inserted into a standard bacterial cloning vector, e.g. Invitrogen Corporation, Carlsbad, CA The construct is then cloned into a TOPO® vector available from the University of California, San Diego, CA. The DNA is sequenced by standard methods. The fragments are 80-90 base pairs long and are linked together. Several of these constructs, consisting of ∼6 fragments, i.e., fragments of approximately 500 base pairs, were used to generate the desired The entire sequence is prepared so that it is represented in a series of plasmid constructs. The inserts in the vectors are cut with the appropriate restriction enzymes and ligated together to form the final construct. The final construct is then cloned into a standard bacterial cloning vector. Additional methods will be readily apparent to those skilled in the art. Gene synthesis is readily available commercially.

[0109] "Modified" refers to, for example, non-viral delivery systems (e.g., RNA-based systems, naked DNA or to generate viral vectors in packaging host cells and / or for delivery to a host cell in a subject. The nucleic acid sequence encoding the protein is assembled, and the TPP1 sequence carried on the element is transfected into the host. Any suitable genetic element to be transferred into the cell, e.g., naked DNA, phage, This means placing the gene in a nucleotide sequence, such as a transposon, cosmid, episome, etc. The genetic element is a plasmid. Methods are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic Techniques include, for example, Green and Sambrook, Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Press, Cold Spring Harbor, NY (201 See 2).

[0110] As used herein, the term "host cell" refers to a cell in which a recombinant AAV is grown from a production plasmid. Alternatively, the term "host cell" may refer to a packaging cell line that produces the coding sequence. It can refer to any target cell in which it is desired to express a sequence. Thus, "host cell" The method may be carried out by any means, such as electroporation, calcium phosphate precipitation, microinjection, or the like. injection, transformation, viral infection, transfection, liposome delivery, membrane Fusion technology, high-speed DNA-coated pellets, viral infection and protoplast fusion a prokaryotic or eukaryotic cell that contains foreign or heterologous DNA introduced into the cell by In certain embodiments herein, the term "host cell" refers to a cell in which a viral vector or a recombinant The term "cells" refers to cells used to produce and package recombinant viruses. In embodiments, the term "host cell" refers to a cell that is capable of in vitro evaluation of the compositions described herein. In yet another embodiment, the term refers to the culture of CNS cells of various mammalian species for the purpose of generating a culture of CNS cells. "Host cells" refers to brain cells of a subject being treated in vivo for Batten disease. Such host cells include the ependyma, i.e., the epithelial lining of the ventricular system, Other host cells include neurons, astrocytes, and epithelial cells of the CNS. These include dendritic cells, and microglia.

[0111] As used herein, the term "treatment" or "treating" refers to the treatment of Batten disease. one or more of the compounds or The term "treatment" is defined to encompass administering a composition to a subject. reducing the onset or progression of neuronal ceroid lipofuscinosis (NCL) in a subject receiving the compound; Prevention of disease, reduction of the severity of disease symptoms, or slowing their progression, including the progression of blindness; Eliminating disease symptoms, delaying the onset of disease, or monitoring disease progression or the effectiveness of treatment The tag may include one or more of the following:

[0112] In one embodiment, the nucleic acid sequence encoding TPP1 has a tag polypeptide covalently attached thereto. The tag polypeptide further includes, but is not limited to, a nucleic acid encoding a myc tag. Polypeptide, glutathione-S-transferase tagged polypeptide, green fluorescent protein Protein tag polypeptide, myc-pyruvate kinase tag polypeptide, His6 tag Polypeptide, influenza virus hemagglutinin tag polypeptide, flag tag polypeptide peptides, and maltose binding protein tag polypeptides. You can also select from the tags.

[0113] In another embodiment, an expression cassette is provided comprising a nucleic acid sequence encoding TPP1. In one embodiment, the sequence is a codon-optimized sequence. The amino acid sequence is SEQ ID NO: 3 which encodes human TPP1.

[0114] As used herein, an "expression cassette" refers to a sequence encoding a TPP1 protein. refers to a nucleic acid molecule that contains a promoter and may contain other regulatory sequences therefor, The vector is packaged into the capsid of a viral vector (e.g., a viral particle) and Generally, such expression cassettes for generating viral vectors are The viral genome packaging signal and other expression control sequences, such as those described herein, The vector comprises a CLN2 sequence described herein, flanked by a sequence of the CLN2 gene. For example, an AAV viral vector In the case of , the packaging signal is the 5' inverted terminal repeat (ITR) and the 3' ITR. When packaged into an AAV capsid, the ITRs in combination with the expression cassette are In the literature, it may be referred to as the "recombinant AAV (rAAV) genome" or "vector genome." In embodiments, the expression cassette comprises a codon-optimized nucleic acid sequence encoding a TPP1 protein. In one embodiment, the cassette comprises a codon encoding TPP1 in a host cell. codon-optimized CLN2 operably associated with an expression control sequence that directs expression of the optimized nucleic acid sequence; to provide.

[0115] In another embodiment, an expression cassette for use in an AAV vector is provided. In embodiments, the AAV expression cassette comprises at least one AAV inverted 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 are codon-optimized nucleic acid sequences encoding TPP1. and optionally, the expression of a codon-optimized nucleic acid sequence encoding TPP1 in a host cell. Thus, as described herein, AAV expression cassettes contain additional sequences that direct expression. The kit is flanked at its 5' end by a 5' AAV inverted terminal repeat (ITR) sequence and at its 3' end by a 3' AAV inverted terminal repeat (ITR). 'It is meant to represent the expression cassette described above flanked by AAV ITRs. The rAAV genome contains expression cassettes in the AAV viral particles, namely the AAV 5' and 3' The AAV ITR contains the minimal sequence required for packaging into the ITR. These ITRs may be obtained from any AAV ITR sequence as described herein. the ITRs are of the same AAV origin as the capsid used in the resulting recombinant AAV, or or ITRs of different AAV origin (to generate AAV pseudotypes). In one embodiment, for convenience and to expedite regulatory approval, AAV 2 or a deleted version thereof (ΔITR). ITRs from other AAV sources may also be selected. If the source of the ITRs is from AAV2, If the AAV capsid is derived from another AAV source, the resulting vector can be pseudotyped. Generally, the AAV vector genome consists of the AAV 5'ITR, TPP1 gene, and The AAV 3'ITR contains the AAV 3'ITR and any regulatory sequences. Other configurations of these elements may also be suitable. D sequence and terminal release site (trs) A shortened version of the 5' ITR, termed ΔITR, has been described in which the 5' ITR has been deleted. The full-length AAV 5' and 3' ITRs are used. Each rAAV genome is then inserted into the production platform. It can be introduced into smids.

[0116] As used herein, the terms "regulatory sequence," "transcriptional regulatory sequence," or "expression regulatory sequence" are used interchangeably. The "sequence" refers to a DNA sequence such as an initiator sequence, an enhancer sequence, and a promoter sequence. refers to sequences that encode proteins to which they are operably linked. Induce, repress, or regulate transcription of a nucleic acid sequence.

[0117] As used herein, the terms "operably linked" or "operably associated" " is an expression regulator contiguous with the nucleic acid sequence encoding TPP1 for regulating transcription and expression. It refers to both nodal sequences and / or trans- or remotely acting expression control sequences.

[0118] In one aspect, a vector is provided that includes any of the expression cassettes described herein. As described herein, such vectors can be plasmids of various origins, As further described herein, specific embodiments for the generation of recombinant replication-defective viruses include: It is useful in such situations.

[0119] As used herein, a "vector" refers to a vector containing a foreign or heterologous or modified nucleic acid. A nucleic acid molecule into which a transgene can be inserted and which can then be introduced into a suitable host cell. Vectors preferably contain one or more origins of replication and a site into which recombinant DNA can be inserted. Vectors often have one or more sites that allow the vector to be transduced from vector-free cells. The vector has a means for selecting cells containing the vector, e.g., the vector is drug-resistant. Common vectors include plasmids, viral genomes, and and (mainly in yeast and bacteria) "artificial chromosomes." It shall be recorded in the book.

[0120] In one embodiment, the vector is a nucleic acid molecule, e.g., a micelle, a liposome, a cationic lipid-nucleic acid combination, or the like. compositions, poly-glycan compositions and other polymer, lipid and / or cholesterol-based nucleic acids Various compositions and nanoparticles can be bound to the nanoparticles, including complexes and other constructs as described herein. non-viral plasmids, e.g., "naked DNA," containing the described expression cassettes, , "naked plasmid DNA", RNA, and mRNA. See, for example, X. Su et al. al,Mol.Pharmaceutics,2011,8(3),pp774-787 ;Web publication: March 21, 2011; WO2013 / 182683, WO201 See WO 2012 / 170930 and WO 2012 / 170930 (all of which are incorporated by reference). Such non-viral TPP1 vectors are described herein. The viral vector or non-viral vector may be administered by the route described in Formulated with a physiologically acceptable carrier for use in gene transfer and gene therapy applications. It can be formulated into a formulation.

[0121] In another embodiment, the vector is a viral vector comprising an expression cassette described herein. A "viral vector" is a vector carrying an exogenous or heterologous CLN2 transgene. In one embodiment, the expression cassettes described herein are defined as replication-deficient viruses. may be engineered onto a plasmid used for drug delivery or viral vector production. Suitable viral vectors are preferably replication-deficient and target brain cells. The viral vector is selected from adenovirus, herpesvirus, and Genes including, but not limited to, tiviruses; retroviruses; parvoviruses, etc. Any virus suitable for treatment may be included. However, for ease of understanding, Nose-associated viruses are referred to herein as exemplary viral vectors.

[0122] "Replication-defective virus" or "viral vector" means an expression cassette containing a gene of interest. Synthetic or recombinant proteins packaged in viral capsids or envelopes refers to the type of virus particle, also packaged within the viral capsid or envelope Any viral genomic sequences that are replicated are replication-deficient; i.e., they are not replicable in progeny. They are unable to produce virions but retain the ability to infect target cells. In this form, the genome of a viral vector does not contain genes encoding enzymes necessary for replication. The transgene of interest is flanked by signals required for amplification and packaging of the artificial genome. This genome can be engineered to be "gutless" and contain only The gene may be supplied during production, thus allowing the virus to replicate in the presence of viral enzymes necessary for replication. Outside this environment, replication and infection by progeny virions cannot occur, making this an ideal candidate for use in gene therapy. It is considered safe for use.

[0123] In another embodiment, a recombinant adeno-associated virus (rAAV) vector is provided. AV comprises an AAV capsid with a vector genome packaged within it.

[0124] The vector genome, in one embodiment, comprises: (a) AAV 5' inverted terminal repeat (ITR) sequences; (b) a promoter; (c) a coding sequence encoding human TPP1; and (d) AAV 3 In another embodiment, the vector genome comprises an expression cassette described herein. In one embodiment, the CLN2 sequence encodes the full-length TPP1 protein. In an embodiment, the TPP1 sequence is the protein sequence of SEQ ID NO: 1. , the coding sequence is SEQ ID NO: 3 or a variant thereof.

[0125] Adeno-associated virus (AAV), a member of the parvovirus family, is 4.7 Small, non-enveloped shouji with a single-stranded linear DNA genome ranging from kilobases (kb) to 6 kb It is a decahedral virus. Known AAV serotypes include AAV1, AAV2, AAV3, and AAV4. AV4, AAV5, AAV6, AAV7, AAV8, AAV9, etc. ITR or Other AAV components can be readily isolated or synthesized from AAV using techniques available to those skilled in the art. Such AAVs may be designed from academic, commercial, or public sources (e.g., A American Type Culture Collection,Manassas Alternatively, AAV sequences may be isolated, designed, or obtained from the literature. or databases, e.g., GenBank, PubMed, etc. By reference to the published sequences, it is possible to design, by synthetic means or other suitable means, AAV viruses may be engineered by conventional molecular biology techniques, thereby , cell-specific delivery of nucleic acid sequences, minimization of immunogenicity, tuning of stability and particle lifetime, efficient disaggregation These particles can be optimized for solution, precise delivery to the nucleus, etc.

[0126] Fragments of AAV are readily available for use in a variety of vector systems and host cells. AAV fragments include vp1, vp2, vp3, and the cap protein containing the hypervariable region; rep proteins, including ep78, rep68, rep52, and rep40; and The sequences encoding these proteins include those fragments that can be used alone or in combination with other A in combination with AV serotype sequences or fragments, or with other AAV sequences or non-AAV sequences As used herein, the term "antibody" may be used in combination with elements derived from virus sequences. Artificial AAV serotypes include, but are not limited to, AAVs with non-native capsid proteins. The novel AAV sequences of the present invention (e.g., fragments of the vp1 capsid protein) are , another AAV serotype (known or novel), non-adjacent parts of the same AAV serotype, non-AAV viruses and in combination with heterologous sequences that may be obtained from viral or non-viral sources. Such artificial capsids may be generated by any suitable technique. It is not limited to chimeric AAV capsids, recombinant AAV capsids, or "humanized" AAV capsids. In one embodiment, the vector may be an AAV9 capsid and / or an AV capsid. or rep sequences. See issue 1.

[0127] In one embodiment, the AAV9 characterized by the amino acid sequence of SEQ ID NO:6 is described herein. and providing an AAV vector carrying a capsid and expressing it in a patient in need thereof. Classical late-infantile neuronal ceroid lipofuscinosis 2(C) is under the control of regulatory sequences that direct Nucleic acids encoding the LN2 gene are provided herein.

[0128] As used herein, "AAV9 capsid" refers to a variant of SEQ ID NO:6. Approximately 60 possible variants are commonly expressed as alternative splicing variants that result in The present invention is characterized by DNAse-resistant particles, which are aggregates of mutant proteins (vp). See also Genbank Accession No. AAS99264.1, which is incorporated herein by reference. See also WO2005 / 033321 and WO2005 / 06111. "AAV9 variants" include, for example, those described in WO2016 / 049230, US8,927, 514, US2015 / 0344911, and Varian, as described in US8,734,809 The amino acid sequence is reproduced in SEQ ID NO: 6, and the coding sequence is In one embodiment, the AAV9 capsid is represented by SEQ ID NO: 7. the encoded capsid, or at least about 90%, 95%, 95%, 98%, or have sequences that share 99% identity.

[0129] The largest protein, vp1, generally corresponds to the full length of the amino acid sequence of SEQ ID NO:6. (aa 1-736 of SEQ ID NO: 6). In a specific embodiment, the AAV9 vp2 protein is The protein has the amino acid sequence of 138 to 736 of SEQ ID NO: 6. In certain embodiments, AAV9 vp3 has the amino acid sequence of 203 to 736 of SEQ ID NO: 6. In some forms, the vp1, 2, or 3 protein may have truncations ( For example, one or more amino acids at the N- or C-terminus. The AAV9 capsid contains approximately 60 vp1, vp2, and vp3 are contained within the assembled capsid. There are about 1 vp, about 1 vp2, and about 10 to 20 vp3 in the nucleus. The ratio may vary depending on the production system used. In certain embodiments, vp2 is absent. Modified AAV9 capsids may be generated.

[0130] This AA contains DNA (genomic or cDNA) or RNA (e.g., mRNA). It is within the skill of the art to design a nucleic acid sequence encoding a V9 capsid. In certain embodiments, the nucleic acid sequence encoding the AAV9 vp1 capsid protein is , provided in SEQ ID NO: 7. In another embodiment, 70% to 99.9% of the The identical nucleic acid sequence may be selected to express the AAV9 capsid. In some embodiments, the nucleic acid sequence is at least about 75% identical to SEQ ID NO:7, at least 80% identical to SEQ ID NO:7, Identical, at least 85%, at least 90%, at least 95%, at least 97% identical , or at least 99% to 99.9% identical.

[0131] As used herein, the term "clade" in reference to a group of AAVs refers to an AAV v Based on the alignment of the p1 amino acid sequences, a few Bootstrap values ​​of at least 75% (of at least 1000 replicates) and less than 0.05 Refers to groups of AAVs that are phylogenetically related to each other, as determined by Poisson-corrected distance measures. Neighbor-joining algorithms are described in the literature, e.g., M. Nei and S .Kumar,Molecular Evolution and Phylogene tics,Oxford University Press,New York(20 00). A computer that can be used to implement this algorithm For example, the MEGA v2.1 program is a modified N Implement the ei-Gojobori method. These technologies and computer programs, Using the sequences of the AAV vp1 capsid protein and the AAV vp2 capsid protein, one skilled in the art can identify the selected AA V may be included in one of the clades identified herein or in another clade. It can be easily determined whether a species is included in or outside these clades. For example, clades A, B, C, D, E, and F were identified, and novel AAV nucleic acid sequences (GenB Gao, et al., provide accession numbers AY530553 to AY530629. See J Virol, 2004 Jun;78(10):6381-6388. See also WO2005 / 033321. AAV9 is within clade F. Other clade F AAVs include AAVhu31 and AAVhu32. Includes:

[0132] As used herein, with respect to AAV, the term variant refers to a variant of the amino acid sequence or nucleic acid sequences and at least 70%, at least 75%, at least 80%, at least At least 85%, at least 90%, at least 95%, at least 97%, at least 99% or any sequence derived from a known AAV sequence, including sequences that share at least one sequence identity with the AAV sequence. In another embodiment, the AAV capsid is any of the described or This includes variants that may contain up to about 10% mutations relative to the known AAV capsid sequence. That is, the AAV capsid may be any of the capsids provided herein and / or known in the art. Approximately 90% to 99.9% identical, approximately 95% to 99% identical to the AAV capsid In one embodiment, the AAV capsid shares about 97% to about 98% identity with the AAV. shares at least 95% identity with the AAV9 capsid. When determining the percentage of identity, a variable protein (e.g., vp1, vp2, or vp3) In one embodiment, the AAV capsid is vp1. , vp1, or vp2, share at least 95% identity with AAV9.

[0133] As used herein, an "artificial AAV" includes, but is not limited to, a non-naturally occurring capsid. AAV having a selected AAV sequence (e.g., vp1 capsid) fragments of a protein) from different selected AAVs, non-adjacent parts of the same AAV, non-AAV in combination with heterologous sequences that may be obtained from viral or non-viral sources, Such artificial capsids may be generated by any suitable technique. However, pseudotyped AAV, chimeric AAV capsids, recombinant AAV capsids, Alternatively, the capsid of one AAV may be a "humanized" AAV capsid. Pseudotyped vectors with capsid protein substitutions are useful in the present invention. In one embodiment, AAV2 / 9 and AAV2 / rh.10 are exemplary pseudotype vectors. He is a ctor.

[0134] In another embodiment, a self-complementary AAV is used. A "self-complementary AAV" refers to a recombinant AAV. The coding region carried by the AAV nucleic acid sequence is designed to form an intramolecular double-stranded DNA template. This refers to a plasmid or vector that has an expression cassette designed to Rather than waiting for cell-mediated synthesis of the scAAV, the two complementary halves of the scAAV associate and immediately to form a single double-stranded DNA (dsDNA) unit ready for replication and transcription. For example, DM McCarty et al., “Self-complement ary recombinant adeno-associated virus(s) cAAV)vectors promote efficient transduct ion independently of DNA synthesis”,Gene Therapy,(August 2001),Vol 8,Number 16,P See, e.g., U.S. Pat. No. 6,248,125. Self-complementary AAVs are described, for example, in U.S. Pat. Nos. 6,596,535; 7,125,717; and 7,456,683. No. 6,299,133, each of which is incorporated herein by reference in its entirety.

[0135] The term "exogenous" when used to describe a nucleic acid sequence or protein refers to the or protein is not naturally present in the chromosome or in the location where it occurs in the host cell. An exogenous nucleic acid sequence also means that the exogenous nucleic acid sequence is derived from the same host cell or subject and is not inserted into the genome but in a non-native state, e.g., in a different copy number or with different regulatory elements. It also refers to a sequence that is under the control of a transcription factor.

[0136] The term "heterologous" when used to describe a nucleic acid sequence or protein means that the nucleic acid or The protein may be expressed in an organism different from the host cell or subject that expresses it, or in the same organism. It means that the protein or the plasmid, expression cassette, if The term "heterologous" when used with respect to a nucleic acid in a vector means that the protein or The presence of nucleic acids with other sequences or subsequences that are not found in the same relationship to each other in nature. Indicates that.

[0137] In yet another embodiment, an expression cassette comprising any of the expression cassettes described herein. The recombinant AAV genome is generated using the vector.

[0138] In one embodiment, to generate viral vectors and / or to generate, for example, naked D To deliver NAs, phages, transposons, cosmids, episomes, etc. into host cells The expression cassettes described herein can be incorporated into suitable genetic elements (vectors) useful for The selected vector is then inserted into the vector, transferring the CLN2 sequence contained therein. cleavage, electroporation, liposome delivery, membrane fusion technology, high-speed DNA coating Transfection can be performed by any suitable method, including thawing pellets, viral infection, and protoplast fusion. The methods used to generate such constructs are within the skill of those skilled in the art of nucleic acid manipulation. Methods are known and include genetic engineering, recombinant engineering, and synthetic techniques. ook et al,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Press,Cold Sp See Ring Harbor, NY.

[0139] Packaging of expression cassette or rAAV genome or production plasmid into virions In order to express AAV only, the ITRs are required in cis within the same construct as the expression cassette. In one embodiment, the replication (rep) and / or capsid (cap) components The AAV genome is then transfected with the nucleotide sequence of the target gene to generate the AAV vector. The vector is supplied by the plasmid or by a packaging cell line.

[0140] Methods for generating and isolating AAV viral vectors suitable for delivery to a subject are known in the art. These are well known in the art. See, for example, U.S. Pat. No. 7,790,449; U.S. Pat. No. 7,282,199; WO 2003 / 042397;WO2005 / 033321, WO2006 / 110689; and US7588772B2. In one system, a production cell line is transfected with an ITR. A construct encoding the flanked transgene and a construct encoding rep and cap (multiple In the second system, rep and cap are stably supplied. A construct encoding a transgene flanked by ITRs was transiently transfected into a packaging cell line that In certain embodiments, the production cell line or packaging cell is transfected. The cell lines are intended to express the AAV viral vectors described herein in suspension culture as production cell lines or or suspension cells, such as may be produced by growing packaging cell lines. Each of these systems requires the infection of helper adenovirus or herpesvirus. AAV virions are produced in response to rAAV, making it necessary to separate rAAV from contaminating viruses. More recently, helper virus infection has not been required for AAV recovery. A system was developed that incorporates the required helper functions (i.e., adenovirus E1, E2a, VA, and E4 or herpesvirus UL5, UL8, UL52, and UL29, The ribosomal DNA polymerases (e.g., ribosomal DNA polymerases and herpesvirus polymerases) are also supplied in trans by the system. In newer systems, cells are transiently transfected with constructs encoding the necessary helper functions. Helper functions can be provided by infecting Cells can be engineered to stably contain a gene encoding a function, and the expression of that gene can be inhibited. Expression can be regulated at the transcriptional or post-transcriptional level.

[0141] The term "isolated" refers to a material that is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, removing natural polynucleotides present in living animals. A peptide or polypeptide is not isolated, but may contain some or all of the substances that coexist in a natural system. The same polynucleotide or polypeptide isolated from a natural system, even if subsequently Such polynucleotides are isolated even when they are reintroduced into a vector. and / or such polynucleotides or polypeptides may be part of a and such vector or composition may be part of its natural environment. It can still be isolated in that it is free of

[0142] In yet another system, the expression cassette and rep / cap genes flanked by ITRs are expressed in a These are introduced into insect cells by infection with a human virion vector. For a general review of production systems, see, for example, Zhang et al., 2009, "Adenovirus-adeno- associated virus hybrid for large-scale recombinant adeno-associated virus produ ction,” Human Gene Therapy 20:922-929 Methods for making and using these and other AAV production systems are also described in the following U.S. patents: Nos. 5, 13, 149, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 169, 170, 171, 172, 1 9,941;5,741,683;6,057,152;6,204,059;6,26 8,213;6,491,907;6,660,514;6,951,753;7,09 4,604; 7,172,893; 7,201,898; 7,229,823; and 7, 439,065. In general, see, for example, Grieger & Samulski, 2005 ,“Adeno-associated virus as a gene thera py vector: Vector development, production and clinical applications,”Adv.Biochem.E ngin / Biotechnol.99:119-145;Buning et al. ,2008,“Recent developments in adeno-asso mediated virus vector technology,”J.Gene M ed. 10:717-733; and the references cited below are , the entire contents of which are incorporated herein by reference.

[0143] In one aspect, a suspension cell line capable of producing rAAV is described herein as a suspension culture medium. Methods for producing the rAAV described herein are provided, including growing the rAAV in culture.

[0144] In certain embodiments, cells are adapted for suspension culture using serum-free and animal component-free media. In certain embodiments, a suspension cell line is derived from an adherent cell line by inducing the cell line to a suspension cell line. The cell line is a HEK293 suspension cell line.

[0145] The methods used to construct any embodiment of the present invention are well known to those skilled in the art of nucleic acid manipulation. Methods include genetic engineering, recombinant engineering, and synthetic techniques. n and Sambrook et al,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Pr ess, Cold Spring Harbor, NY (2012). Similarly, methods for producing rAAV virions are well known, and the selection of an appropriate method is not a limitation of the present invention. For example, K. Fisher et al. (1993) J. Virol. ., 70:520-532 and U.S. Patent No. 5,478,745.

[0146] "Plasmids" are generally capitalized according to standard naming conventions familiar to those of skill in the art. The following are designated by a lowercase p before and after letters and / or numbers: Many plasmids and other cloning and expression vectors suitable for this purpose are well known, Further, those skilled in the art will be able to identify suitable compounds for use in the present invention. Any number of other plasmids similar to those described herein may be readily constructed. The properties, construction and use of these and other vectors will be readily apparent to those skilled in the art from this disclosure. There will be.

[0147] In one embodiment, the production plasmid is a plasmid described herein or Plasmids described in WO2012 / 158757, which is incorporated herein by reference. A variety of plasmids are publicly available in the art for use in generating rAAV vectors. AAV cap and / or rep proteins are known and useful herein. The production plasmid is cultured in host cells that express each rAAV genome. Rescued and packaged into capsid or envelope proteins This allows infectious virus particles to form.

[0148] In one aspect, a production plasmid is provided comprising the expression cassette described above. In one embodiment, The production plasmid is the plasmid shown in Figure 1B. This plasmid is rAAV-human Used in the example of generating a codon-optimized TPP1 vector. Such a plasmid is Contains the 5' AAV ITR sequence; a selected promoter; a polyA sequence; and a 3' ITR and the plasmid is a nucleotide sequence encoding an intron such as a chicken β-actin intron. An exemplary schematic is shown in Figure 1A. In a further embodiment, the intron sequence The columns are approximately 3 kilobases (kb) to approximately 6 kb, approximately 4.7 kb to approximately 6 kb, and approximately 3 kb to approximately 5. Holds rAAV vector genomes of 5 kb, or approximately 4.7 kb to 5.5 kb in size An example of a production plasmid containing the TPP1 coding sequence can be found in SEQ ID NO: 5. In this embodiment, the production plasmid is modified to optimize the efficiency of vector plasmid production. Such modifications may include adding other neutral sequences or modifying the sequence of the vector plasmid. Examples include the inclusion of a lambda stuffer sequence to adjust the level of supercoiling. Such modifications are contemplated herein. In other embodiments, terminators and Other sequences are included in the plasmid.

[0149] In certain embodiments, rAAV expression cassettes, vectors (such as rAAV vectors), The virus (e.g., rAAV), and / or the production plasmid may contain AAV inverted terminal end sequences, TPP a codon-optimized nucleic acid sequence encoding 1, and / or the encoded tandem in a host cell. In another embodiment, the rAAV expression cassette comprises an expression control sequence that directs expression of the protein. The virus, vector (e.g., rAAV vector), and / or production plasmid are It may further contain one or more of the following: a nucleotide sequence, a Kozak sequence, polyA, a post-transcriptional regulatory element, etc. In one embodiment, the post-transcriptional regulatory element is a woodchuck hepatitis virus (WHP) It is a post-transcriptional regulatory element (WPRE).

[0150] Expression cassettes, vectors and plasmids may be used as described herein, e.g., The method can be optimized for a particular species using techniques known in the art, including optimization. The cassettes, vectors, plasmids and viruses described herein may also contain other components. Another composition component includes a promoter sequence as part of the expression control sequence. In this embodiment, the promoter is cell-specific. The term "cell-specific" refers to the The specific promoters selected for the purpose are optimized for specific cell or tissue types. In one embodiment, the TPP1 gene is capable of directing expression of a TPP1 coding sequence. The promoter is specific for expression of the transgene in the ependyma, the epithelial lining of the ventricular system. In another embodiment, the promoter is a promoter for neurons, astrocytes, oligodendrocytes, or The expression of the IL-111 gene is specific for expression in brain cells selected from the group consisting of myocytes, and microglia. In this case, the promoter may be programmed to add one or more restriction enzyme sites to facilitate cloning. Modify the motor.

[0151] In another embodiment, the promoter is a ubiquitous or constitutive promoter. An example of a promoter has a cytomegalovirus (CMV) enhancer element. Hybrid chicken beta-actin (CBA) promoter, e.g., nt of SEQ ID NO: 5 In another embodiment, the promoter is the CB7 promoter. Other suitable promoters include the human β-actin promoter, the human Elongation factor-1α promoter, cytomegalovirus (CMV) promoter, simian virus 40 promoter and herpes simplex virus thymidine kinase promoter For example, Damdindorj et al. (August 2014) A Comparative Analysis of Constitutive P romoters Located in Adeno-Associated Vir See al Vectors. PLoS ONE 9(8):e106472. Other suitable promoters include viral promoters, constitutive promoters, regulated promoters, and the like. promoters [e.g., WO2011 / 126808 and WO2013 / Alternatively, promoters that respond to physiological cues can be used as described herein. In the expression cassettes, rAAV genomes, vectors, plasmids, and viruses described herein, In one embodiment, the promoter may be In another embodiment, the promoter is 40 bp or less. The promoter length is less than 0 bp. Other promoters may be selected by those skilled in the art.

[0152] In further embodiments, the promoter is an SV40 promoter, dihydrofolate reductase tase promoter, phage λ (PL) promoter, herpes simplex virus (HSV ) promoter, tetracycline-regulated transactivator-responsive promoter ( tet) system, RSV LTR, MoMLV LTR, BIV LTR or HIV LTR Long terminal repeat (LTR) promoters such as R, U3 region of Moloney murine sarcoma virus promoter, granzyme A promoter, regulatory sequences of metallothionein genes (multiple CD34 promoter, CD8 promoter, thymidine kinase (TK) promoter tar, B19 parvovirus promoter, PGK promoter, glucocorticoid promoter Heat shock proteins (HS) such as the promoters of the HSP65 and HSP70 P) promoter, immunoglobulin promoter, MMTV promoter, Rous sarcoma virus RSV promoter, lac promoter, CaMV 35S promoter, Nopaline synthetase promoter, MND promoter, or MNC promoter The promoter sequences are known to those skilled in the art or can be found in the literature or For example, publicly available in databases such as GenBank, PubMed, etc. do.

[0153] In another embodiment, the promoter is an inducible promoter. rapamycin / rapalog promoter, ecdysone promoter, estrogen response resistant promoter, and tetracycline-responsive promoter, or heterodimeric promoter The promoter can be selected from known promoters containing a promoter switch, all of which are incorporated by reference in their entirety. Sochor et al., An Autogenously Regulated Expression System for Gene Th erapeutic Ocular Applications.Scientific Reports, 2015 Nov 24;5:17105 and Daber R, Le wis M.,A novel molecular switch.J Mol Bi ol.2009 Aug 28;391(4):661-70,Epub 2009 J See un 21.

[0154] In other embodiments, the expression cassettes, vectors, plasmids, and viruses described herein The sequence may contain other suitable transcription initiation sequences, transcription termination sequences, enhancer sequences, splicing signals, etc. Efficient RNA processing signals, such as null and polyadenylation (polyA) signals TATA sequence; a sequence that stabilizes cytoplasmic mRNA; a sequence that enhances translation efficiency (i.e., Kozak consensus sequence); introns; sequences that enhance protein stability; and Optionally, it contains sequences that facilitate secretion of the encoded product. , including none, one or more of the elements described herein. good.

[0155] Examples of suitable polyA sequences include, for example, synthetic polyA or bovine growth hormone (bGH). , human growth hormone (hGH), SV40, rabbit β-globin (RGB), or modified In a further embodiment, the poly A is derived from mRGF (mRGF). , having the nucleic acid sequence of nt 33 to 159 of SEQ ID NO:5.

[0156] Examples of suitable enhancers include, for example, a CMV enhancer, an RSV enhancer, Alpha-fetoprotein enhancer, TTR minimal promoter / enhancer, LSP(T H-binding globulin promoter / α1-microglobulin / bikunin enhancer), A PB enhancer, ABPS enhancer, αmik / bik enhancer, TTR enhancer Hanser, en34, ApoE, etc.

[0157] In one embodiment, a Kozak sequence is included upstream of the TPP1 coding sequence to identify the correct start sequence. In another embodiment, the expression cassette contains CBA exon 1 and In one embodiment, the TPP1 coding sequence is a hybrid chicken β It is placed under the control of the actin (CBA) promoter. This promoter is The CMV immediate early enhancer, the proximal chicken β-actin promoter, and the It consists of CBA exon 1 flanked by tron ​​1 sequences.

[0158] In another embodiment, the intron is CBA, human beta globin, IVS2, SV40, b GH, alpha globulin, beta globulin, collagen, ovalbumin, p53, or The fragment is selected from:

[0159] In one embodiment, the expression cassettes, vectors, plasmids and viruses include a 5' ITR, Chicken β-actin (CBA) promoter, CMV enhancer, CBA exon 1 and and intron, human codon-optimized CLN2 sequence, rabbit globin polyA and 3'IT In a further embodiment, the expression cassette comprises nt 1 to 4020 of SEQ ID NO: 8. In yet a further embodiment, the 5' ITR comprises nt 3199 to nt 4000 of SEQ ID NO:5. 3328 nucleic acid sequence, and the 3'ITR is from nt 248 to nt 377 of SEQ ID NO:5. In a further embodiment, the production plasmid has the nucleic acid sequence of SEQ ID NO: 5. This is also shown in Figures 1C to 1E.

[0160] II. Methods and Pharmaceutical Compositions for Treating Batten Disease II-1. Treatment for Batten disease In another aspect, the method for treating Batten disease caused by a defect in the CLN2 gene is and administering to a subject in need thereof a vector encoding TPP1 (r In one embodiment, the subject with Batten disease is treated with a medicament described herein, including delivery of a medicament to the subject (e.g., an AAV). Also provided herein are methods of treating with the rAAV described herein. and administering the rAAV to a subject in need thereof by multiple routes. The present invention provides a method for treating

[0161] In one aspect, the recombinant adeno-associated virus (rAAV) is described herein as a first transfection vector. and administering the compound to a subject in need thereof via a first route and a second route. A method for treating CLN2 Batten disease is provided, wherein the first pathway and the second pathway are central nervous system the first pathway to the central nervous system (CNS), the second pathway to a brain region, The pathway to the spinal cord is the pathway to the spinal cord, and the recombinant adeno-associated virus (rAAV) a capsid and a vector genome packaged therein, , comprising: (a) AAV 5' inverted terminal repeat (ITR) sequences; (b) a promoter; (c ) the CLN2 coding sequence encoding human TPP1; and (d) the AAV 3'ITR.

[0162] In certain embodiments, the brain region may be the intrathecal space that covers the brain. In certain embodiments, the brain region may be a ventricle. In certain embodiments, the brain region may be a cisterna magna. In some embodiments, delivery to a brain region can be delivery to the cerebrospinal fluid (CSF).

[0163] In certain embodiments, the spinal cord region can be the intrathecal space surrounding the spinal cord. In certain embodiments, the spinal region may be the spinal canal. In certain embodiments, the spinal region is the subarachnoid space. In certain embodiments, delivery to the spinal region is delivery to the cerebrospinal fluid (CSF). obtain.

[0164] In certain embodiments, the first route is intracerebroventricular (ICV) or intracisternal (IC) In other embodiments, the first pathway is intracerebral other than intracerebroventricular (ICV) or intracisternal (IC). The route of administration to the area.

[0165] In certain embodiments, the second route is intrathecal (IT-L). In some embodiments, the first route is an administration route to a spinal region other than intrathecal lumbar (IT-L). .

[0166] In certain embodiments, the method includes administering the rAAV to the subject via a third route. the third route being intracerebroventricular (ICV), intracisternal (IC), lumbar intrathecal intravenous, intravascular, intraarterial, intramuscular, intraocular, subcutaneous, and intradermal. In certain embodiments, the third pathway delivers rAAV to the liver. In an embodiment, the third route is intravenous.

[0167] In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises intracerebroventricular (ICV) ) and intrathecal (IT-L) routes to deliver the rAAV to subjects in need thereof. In certain embodiments, the method of treating CLN2 Batten disease in a subject includes co-administering The method involves the intracisternal (IC) and intrathecal lumbar (IT-L) routes of delivery of the rAAV. In certain embodiments, the method comprises co-administering the CLN2 antibody to a subject in need thereof. Treatment of Tutten's disease is available via intraventricular (ICV), intrathecal (IT-L), and intravenous routes. and co-administering the rAAV to a subject in need thereof via a cellular immunoglobulin (mAb). In this state, the method of treating CLN2 Batten disease in a subject includes intracisternal (IC), lumbar intrathecal (I) TL) and co-administering the rAAV to a subject in need thereof via the intravenous route. This includes:

[0168] In another embodiment, recombinant adeno-associated virus (rAAV) is prepared by the first and second pathways. administering to a subject in need thereof a CLN2 batten in a subject via The present invention provides a method for treating a disease, wherein the first pathway is a pathway to the central nervous system (CNS), The second route delivers rAAV outside the CNS and delivers the recombinant adeno-associated virus (rAAV) to the host. AAV (AAV) contains the AAV capsid and the vector genome packaged within it. The vector genome comprises: (a) an AAV 5' inverted terminal repeat (ITR) sequence; (b) (c) a CLN2 coding sequence encoding human TPP1; and (d) an AAV In certain embodiments, the first route is intrathecal (IT-L), intravenous, or intrathecal. In certain embodiments, the second route is intravenous (ICV) or intracisternal (IC). Intravenous, intravascular, intraarterial, intramuscular, intraocular, subcutaneous, and intradermal. In an embodiment, the second route is intravenous.

[0169] In another embodiment, recombinant adeno-associated virus (rAAV) is prepared by the first and second pathways. administering to a subject in need thereof a CLN2 batten in a subject via The present invention provides a method for treating a disease, wherein the first pathway is a pathway to the central nervous system (CNS), The second route delivers rAAV to the liver, and the recombinant adeno-associated virus (rAAV) , comprising an AAV capsid and a vector genome packaged therein, The genome includes: (a) the AAV 5' inverted terminal repeat (ITR) sequence; (b) the promoter sequence; (c) the CLN2 coding sequence encoding human TPP1; and (d) AAV 3′I TR.

[0170] In certain embodiments, the first route is intrathecal (IT-L), intracerebroventricular (ICV), or intracisternal (IC). In other embodiments, the first route is lumbar intrathecal (IT- L), a route of administration to the CNS other than intracerebroventricular (ICV) or intracisternal (IC).

[0171] In certain embodiments, the second route is intravenous, intravascular, intraarterial, intramuscular, intraocular, or cutaneous. In certain embodiments, the second route is selected from the group consisting of intravenous, intravenous, intradermal, and intradermal. In other embodiments, the second route is intravenous, intravascular, intraarterial, intramuscular, or intraocular. , subcutaneous, and intradermal are administration routes that deliver rAAV to the liver.

[0172] In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises administering to the CNS and venous The intravenous route involves co-administering the rAAV to a subject in need thereof. In one embodiment, the method for treating CLN2 Batten disease in a subject includes administering the drug via the intrathecal and intravenous routes. and co-administering the rAAV to a subject in need thereof. The method of treating CLN2 Batten disease in a subject includes intrathecal (IT-L) and intravenous and co-administering the rAAV to a subject in need thereof via a specific route. In embodiments, the method of treating CLN2 Batten disease in a subject includes intracerebroventricular (ICV) and intravenous administration. and co-administering the rAAV to a subject in need thereof via an intravenous route. In certain embodiments, the method of treating CLN2 Batten disease in a subject includes intracisternal (IC) and intravenous administration. and co-administering the rAAV to a subject in need thereof via an intravenous route. In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises lumbar intrathecal (IT- L), intracerebroventricular (ICV) or intracisternal (IC), and via routes other than the intravenous route to the CNS and co-administering the rAAV to a subject in need thereof.

[0173] In certain embodiments, the method of treating CLN2 Batten disease in a subject includes treating CNS and vascular and co-administering the rAAV to a subject in need thereof via an intravenous route. In certain embodiments, the method of treating CLN2 Batten disease in a subject includes intrathecal and intravenous administration. and co-administering the rAAV to a subject in need thereof via an intraluminal route. In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises lumbar intrathecal (IT- L) and co-administering the rAAV to a subject in need thereof via an intravascular route. In certain embodiments, the method for treating CLN2 Batten disease in a subject comprises administering intraventricular ( The rAAV is co-administered to a subject in need thereof via intravenous (ICV) and intravascular routes. In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises intracisternal administration. and co-administering the rAAV to a subject in need thereof via intravenous (IC) and intravascular routes. In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises administering to a subject a lumbar spine or pelvic floor muscle. Routes other than intrathecal (IT-L), intraventricular (ICV) or intracisternal (IC), and intravascular routes The method includes co-administering the rAAV to the CNS via a cytotoxic T cell antigen (CTA) to a subject in need thereof.

[0174] In certain embodiments, the method of treating CLN2 Batten disease in a subject includes treating the CNS and arteries and co-administering the rAAV to a subject in need thereof via an intravenous route. In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises intrathecal and intravenous administration. and co-administering the rAAV to a subject in need thereof via an intravenous route. In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises lumbar intrathecal (IT- L) and co-administration of the rAAV to a subject in need thereof via the intra-arterial route. In certain embodiments, the method for treating CLN2 Batten disease in a subject comprises administering intraventricular ( The rAAV is co-administered to a subject in need thereof via intravenous (ICV) and intra-arterial routes. In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises intracisternal administration. (IC) and intra-arterial routes to administer the rAAV to a subject in need thereof. In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises administering to a subject a lumbar spine or pelvic floor muscle. Routes other than intrathecal (IT-L), intraventricular (ICV) or intracisternal (IC), and intra-arterial routes The method includes co-administering the rAAV to the CNS via a cytotoxic T cell antigen (CTA) to a subject in need thereof.

[0175] In certain embodiments, the method of treating CLN2 Batten disease in a subject includes treating CNS and muscle and co-administering the rAAV to a subject in need thereof via an intravenous route. In certain embodiments, the method of treating CLN2 Batten disease in a subject includes intrathecal and intramuscular injection. and co-administering the rAAV to a subject in need thereof via the intramuscular route. In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises lumbar intrathecal (IT- L) and co-administering the rAAV to a subject in need thereof via the intramuscular route. In certain embodiments, the method for treating CLN2 Batten disease in a subject comprises administering intraventricular ( The rAAV is co-administered to a subject in need thereof via intravenous (ICV) and intramuscular routes. In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises intracisternal administration. and co-administering the rAAV to a subject in need thereof via intravenous (IC) and intramuscular routes. In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises administering to a subject a lumbar spine or pelvic floor muscle. Routes other than intrathecal (IT-L), intracerebroventricular (ICV) or intracisternal (IC), and intramuscular routes The method includes co-administering the rAAV to the CNS via a cytotoxic T cell antigen (CTA) to a subject in need thereof.

[0176] In certain embodiments, the method of treating CLN2 Batten disease in a subject includes administering to the CNS and intraocular and co-administering the rAAV to a subject in need thereof via a route to In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises intrathecal and intraocular and co-administering the rAAV to a subject in need thereof via a specific route. In embodiments, the method of treating CLN2 Batten disease in a subject comprises intrathecal lumbar (IT-L) administration of and co-administering the rAAV via an intraocular route to a subject in need thereof. In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises intracerebroventricular (ICV) and co-administration of the rAAV to a subject in need thereof via the intraocular route. In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises intracisternal (IC) and co-administering the rAAV via an intraocular route to a subject in need thereof. In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises administering lumbar intrathecal (I TL), intracerebroventricular (ICV) or intracisternal (IC), and CN via routes other than the intraocular route S includes co-administering the rAAV to a subject in need thereof.

[0177] In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises administering to the CNS and subcutaneous and co-administering the rAAV to a subject in need thereof via a route to In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises intrathecal and subcutaneous administration. and co-administering the rAAV to a subject in need thereof via a specific route. In embodiments, the method of treating CLN2 Batten disease in a subject comprises intrathecal lumbar (IT-L) administration of and co-administering the rAAV via a subcutaneous route to a subject in need thereof. In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises intracerebroventricular (ICV) and co-administration of the rAAV to a subject in need thereof via the subcutaneous route. In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises intracisternal (IC) and co-administering the rAAV via a subcutaneous route to a subject in need thereof. In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises administering lumbar intrathecal (I TL), intracerebroventricular (ICV) or intracisternal (IC), and CN via routes other than the subcutaneous route S includes co-administering the rAAV to a subject in need thereof.

[0178] In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises administering to the CNS and intradermal and co-administering the rAAV to a subject in need thereof via a route to In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises intrathecal and intradermal administration. and co-administering the rAAV to a subject in need thereof via a specific route. In embodiments, the method of treating CLN2 Batten disease in a subject comprises intrathecal lumbar (IT-L) administration of and co-administering the rAAV via an intradermal route to a subject in need thereof. In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises intracerebroventricular (ICV) and co-administration of the rAAV to a subject in need thereof via the intradermal route. In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises intracisternal (IC) and co-administering the rAAV via an intradermal route to a subject in need thereof. In certain embodiments, the method of treating CLN2 Batten disease in a subject comprises administering lumbar intrathecal (I TL), intracerebroventricular (ICV) or intracisternal (IC), and CN via routes other than the intradermal route S includes co-administering the rAAV to a subject in need thereof.

[0179] In certain embodiments, the methods for treating CLN2 Batten disease provided herein include treating the second and administering the rAAV via the first route simultaneously with administering the rAAV via the second route. The method may include administering AV.

[0180] In certain embodiments, the methods for treating CLN2 Batten disease provided herein include treating the second administering the rAAV via the first route before administering the rAAV via the second route In certain embodiments, the CLN2 Batten disease vectors provided herein may include administering the vector to a patient. The treatment method comprises administering the rAAV via the second route, followed by administration of the rAAV via the first route. The method may include administering the rAAV to a patient.

[0181] In certain embodiments, administration of the rAAV via the first route and the second route The interval between administration of the rAAV via the 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 1 hour 1 hour, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 2 weeks, about 3 weeks, about 4 weeks About 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, or more It may be more than that.

[0182] In certain embodiments, administration of the rAAV via the first route and the second route The interval between administration of the rAAV via the IV injection is 0.5 hours, 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours. Hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, about 1 Day, 2nd, 3rd, 4th, 5th, 6th, 1 week, 8th, 9th, 10th, 11th, 12th, 1 3 days, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or longer It may be above.

[0183] In certain embodiments, the methods for treating CLN2 Batten disease provided herein include administering to the subject In certain embodiments, the methods provided herein may result in increased TPP1 activity in the spinal cord of a subject. The method for treating CLN2 Batten disease comprises administering to the spinal cord of a subject a gene encoding a gene encoding a CLN2 Batten disease gene in the spinal cord of a second subject. at least 2%, 3%, 5%, 6%, 7%, 8%, 9%, or 10% higher than the reference TPP1 activity 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% higher TPP1 activity, and the reference TPP1 activity in the spinal cord is The method is performed when two subjects have not received treatment using the method, and the second subject has: In certain embodiments, the subject may be the same as or different from the subject. The method for treating CLN2 Batten disease comprises administering to the spinal cord of a subject a gene encoding ... Compared to the reference TPP1 activity, 2%, 3%, 5%, 6%, 7%, 8%, 9%, 10%, 12 %, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% and the reference TPP1 activity in the spinal cord is higher than that of the second subject. The second subject is measured in the absence of treatment using the method, and the second subject has the same are the same or different.

[0184] In certain embodiments, the methods for treating CLN2 Batten disease provided herein include administering to the subject In certain embodiments, the CL provided herein may result in an increase in hepatic TPP1 activity. The method for treating N2 Batten disease comprises reducing the liver TPP1 activity of at least 2% of the second subject relative to a reference liver TPP1 activity. %, 3%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% greater hepatic TPP1 activity in the subject and the reference hepatic TPP1 activity is determined by comparing the second subject to the reference hepatic TPP1 activity after the second subject has been treated using the method. The second subject may be the same as or different from the first subject. In certain embodiments, the methods for treating CLN2 Batten disease provided herein include In the elephant spinal cord, 2%, 3%, and 4% TPP1 activity was observed compared to the reference TPP1 activity in the spinal cord of the second subject. 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 3 0%, 35%, 40%, 45%, or 50% higher TPP1 activity in the spinal cord The reference TPP1 activity in the second subject is the same as that in the first subject if the second subject has not been treated using the method. The second subject may be the same as or different from the first subject.

[0185] In certain embodiments, the methods for treating CLN2 Batten disease provided herein include administering to the subject In certain embodiments, the C provided herein may result in an increase in serum TPP1 activity. The method of treating LN2 Batten disease comprises at least Also 2%, 3%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20 %, 25%, 30%, 35%, 40%, 45%, or 50% higher serum TPP in the subject 1 activity, and the reference serum TPP1 activity can be determined by comparing the serum TPP1 activity with the serum TPP1 activity of the second subject after treatment using the method. The second subject may be the same or different from the first subject, and the second subject may be measured in the absence of treatment. In certain embodiments, the methods for treating CLN2 Batten disease provided herein include , 2%, 3%, 5%, 6%, 7%, 8%, compared to the reference serum TPP1 activity of the second subject. 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 4 5%, or 50% higher serum TPP1 activity in the subject, and The efficacy is measured when the second subject is not treated using the method, and the efficacy is measured when the second subject is not treated using the method. The two objects may be the same as or different from the object in question.

[0186] In certain embodiments, the methods for treating CLN2 Batten disease provided herein include administering to the subject In certain embodiments, the methods provided herein may result in a reduction in microglial activity within the cortex of a subject. The method for treating CLN2 Batten disease includes administering to the cortex of a subject a gene encoding a CLN2-containing antibody, the gene encoding a CLN2-containing antibody, and the method further comprises administering to the cortex of a second subject a gene encoding a CLN2-containing antibody. At least 2%, 3%, 5%, 6%, 7%, 8% higher than reference microglial activity in ,9%,10%,12%,15%,17%,20%,25%,30%,35%,40%, This may result in 45% or 50% less microglial activity than the reference microglial activity in the cortex. The activity is measured when the second subject is not being treated using the method, The second subject is the same as or different from the subject. The method for treating CLN2 Batten disease provided herein comprises the step of: 2%, 3%, 5%, 6%, 7%, and 8% compared to reference microglial activity in the elephant cortex. ,9%,10%,12%,15%,17%,20%,25%,30%,35%,40%, This may result in 45% or 50% less microglial activity than the reference microglial activity in the cortex. The activity is measured when the second subject is not being treated using the method, The second subject may be the same as or different from the subject.

[0187] In certain embodiments, the methods for treating CLN2 Batten disease provided herein include administering to the subject In certain embodiments, the compounds provided herein may result in increased TPP1 activity in the brain. The method of treating CLN2 Batten disease may further reduce TPP1 activity in the brain of a second subject relative to a reference TPP1 activity in the brain of the second subject. At most 2%, 3%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% higher in the subject's brain and a reference TPP1 activity in the brain can be determined by comparing the TPP1 activity in the brain of the second subject using the method. The second subject is the same as the first subject, but is not receiving the treatment. In certain embodiments, the treatment of CLN2 Batten disease provided herein is The treatment method reduces TPP1 activity by 2%, 3%, 5%, 6%, 7%, or 8% relative to the reference TPP1 activity in the brain of the second subject. %, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% greater TPP1 activity in the subject's brain, The reference TPP1 activity is measured when the second subject is not treated using the method. A second object is defined, and the second object may be the same as or different from the first object.

[0188] In certain embodiments, the rAAV is administered in a therapeutically effective amount.

[0189] In certain embodiments, the subject is a human.

[0190] In certain embodiments, the coding sequence of (c) is a codon-optimized human CLN2, is at least 70% identical to the native human coding sequence of SEQ ID NO: 2. In one embodiment, the coding sequence of (c) is SEQ ID NO:3.

[0191] In certain embodiments, the rAAV capsid is AAV9 or a variant thereof.

[0192] In a specific embodiment, the promoter is the chicken beta actin (CBA) promoter. In certain embodiments, the promoter comprises a CBA promoter sequence and a cytomegalovirus promoter sequence. It is a hybrid promoter containing a nucleotide enhancer element.

[0193] In certain embodiments, the AAV 5'ITR and / or the AAV 3'ITR are 2 is derived from

[0194] In certain embodiments, the vector genome further comprises polyA. The polyA may be synthetic polyA or may be derived from bovine growth hormone (bGH), human growth hormone (HGH), or other suitable polyA. (hGH), SV40, rabbit β-globin (RGB), or modified RGB (mRGB) This is the origin.

[0195] In certain embodiments, the vector genome further comprises an intron. Introns include CBA, human beta globin, IVS2, SV40, bGH, and alpha globin. These may be derived from leukocyte antigens, beta-globulin, collagen, ovalbumin, or p53.

[0196] In certain embodiments, the vector genome further comprises an enhancer. So, the enhancers are CMV enhancer, RSV enhancer, and APB enhancer. , ABPS enhancer, αmic / bik enhancer, TTR enhancer, en3 4. ApoE.

[0197] In certain embodiments, the vector genome is between about 3 kilobases and about 5.5 kilobases in size. In certain embodiments, the vector genome is about 4 kilobases in size.

[0198] In certain embodiments, suspension cell lines capable of producing rAAV are grown in suspension culture. In certain embodiments, the rAAV is produced using a method comprising: The cell line is a HEK293 suspension cell line.

[0199] In certain embodiments of the invention, the subject has neuronal ceroid lipofuscinosis (NCL). , which the components, compositions, and methods of the present invention are designed to treat. As used herein, the term "subject" refers to a human, veterinary animal, or domestic animal, means mammals, including breeding animals or pets, and animals commonly used in clinical research. In embodiments, the subject of these methods and compositions is a human. Examples include, but are not limited to, mice, rats, dogs, cats, pigs, cows, sheep, and non-human primates. As used herein, the term "subject" is used interchangeably with "patient." It is used.

[0200] Neuronal ceroid lipofuscinosis (NCL) is a condition characterized by progressive intellectual and motor deterioration, It is a group of inherited neurodegenerative lysosomal storage disorders characterized by severe visual impairment, severe cognitive impairment, and early death. Loss of function is characteristic of most forms. Clinical phenotypes have traditionally been divided into infantile, late infantile-onset, and early-onset forms. Childhood, adult, and northern epilepsy (also known as progressive epilepsy with mental retardation [EPMR]) It has been characterized according to the age of onset and the order of appearance of the clinical features of the idiopathic encephalopathy (known as idiopathic encephalopathy). However, genetic and allelic heterogeneity exists; both the causative gene and the age of onset vary. New nomenclature and classification systems have been proposed and developed to take into account the For example, classic late-infantile CLN2 disease. First symptoms usually appear between the ages of 2 and 4 years. manifests, usually beginning with epilepsy, followed by developmental regression, myoclonic ataxia Visual impairment usually appears between the ages of 4 and 6 and progresses rapidly to impaired perception of light and dark. Life expectancy ranges from age 6 to early teens. The term "Batten disease" is used to refer to CLN2 disease, which is the same as "NCL." It is used in this sense.

[0201] As used herein, the term "treatment" or "treating" refers to the treatment of Batten disease. one or more of the compounds or The term "treatment" is defined to encompass administering a composition to a subject. reducing the onset or progression of neuronal ceroid lipofuscinosis (NCL) in a subject receiving the compound; Prevention of disease, reduction of the severity of disease symptoms, or slowing their progression, including the progression of blindness; Eliminating disease symptoms, delaying the onset of disease, or monitoring disease progression or the effectiveness of treatment The tag may include one or more of the following:

[0202] As used in the method, "administration" refers to the administration of target selected cells characterized by a defect in the CLN2 gene. In one embodiment, the method includes delivering the composition to the patient by intrathecal injection. In another embodiment, an ICV injection is used to deliver the substance to the subject. In one embodiment, the method comprises administering an intrathecal (IT-L) injection into the subject. Delivering the composition via intracisternal (IC) injection (i.e., after image guidance into the cisterna magna) Intrathecal delivery via subcapsular puncture). As used herein, the term intrathecal refers to In some embodiments, this may refer to intracisternal injection. In yet another method, intravascular injection may be used. In another embodiment, intramuscular injection is used. Other administration methods may be selected.

[0203] "Administration" or "route of administration" refers to the administration of a compound, in the presence or absence of a pharmaceutical carrier or excipient. The administration of the compositions described herein to a subject is also advantageous. Optionally, the administration routes may be combined. In some embodiments, the administration is repeated periodically. The composition may be administered by any suitable route or combination of different routes as required. In some embodiments, direct delivery to the brain (optionally by intrathecal, intracisternal, ICV or IT-L injection), or by systemic routes, e.g., intravenous injection into the blood. Delivery by intraductal, intraarterial, intraocular, intravenous, intramuscular, subcutaneous, intradermal, and other parenteral routes of administration The nucleic acid molecules, expression cassettes and / or vectors described herein are used in a single The composition may be delivered in a composition or compositions. Optionally, two or more different AAVs or One or more viruses may be delivered [see, e.g., WO2011 / 126808 and WO2 In another embodiment, the plurality of viruses are different viruses. Defective viruses (e.g., AAV and adenovirus) alone or in combination with proteins As used herein, the term "intrathecal delivery" or "intrathecal infusion" may include a combination of "Administered" refers to administering a drug to the spinal canal, more specifically the arachnoid membrane, so that the drug reaches the cerebrospinal fluid (CSF). Intrathecal delivery refers to the administration of drugs by injection into the subclavian cavity. (including intraventricular (ICV)), suboccipital / intracisternal, and / or C1-2 puncture may be included. For example, the substance may be introduced by lumbar puncture for diffusion throughout the subarachnoid space. In another example, the injection may be intracisternal.

[0204] As used herein, the term "intracisternal delivery" or "intracisternal administration" refers to administration of a substance to the brain via the cisterna magna of the cerebellum. Directly into the spinal fluid, more specifically via suboccipital puncture or direct injection into the cisterna magna This refers to the route of administration of drugs by means of a catheter or through a permanently placed tube. Devices useful for delivering the compositions described herein to the cerebrospinal fluid are disclosed in US Pat. No. 6,399,423, which is incorporated herein by reference. This application is described in PCT / US2017 / 16133.

[0205] II-2. Pharmaceutical Compositions In another aspect, pharmaceutical compositions are also provided herein.

[0206] In certain embodiments, the pharmaceutical compositions provided herein comprise: (a) a recombinant adeno-associated virus (RAV) (b) sodium chloride, (c) magnesium chloride, (d) potassium chloride (e) dextrose, (f) poloxamer 188, (g) monobasic sodium phosphate, and (h) dibasic sodium phosphate. In certain embodiments, the pharmaceutical composition comprises calcium chloride. It further contains sodium.

[0207] In certain embodiments, the rAAV in the pharmaceutical composition is any rAAV known in the art. In certain embodiments, the rAAV in the pharmaceutical composition may be any of the compounds described in the following patent applications: PCT / US2017 / 027650 (published as International Publication No. WO2017 / 181021) , PCT / US2018 / 027568 (International Publication No. WO2018 / 191666) PCT / US2018 / 015910 (International Publication No. WO2018 / 14444 1), PCT / US2018 / 052855 (International Publication No. WO2019 / 0 No. 67540), PCT / US2019 / 042205, PCT / US201 9 / 043631, WO2019079494A1, WO2019164854A1, W<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> O2019079496A2, US20190211091A1, US20190387<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 77A1, US2018289839A1, US2019127455A1, KR201<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 60010526A、KR20190086503A、TW201903146A、WO<h2 style=";text-align:left;direction:ltr"> <h2 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WO02100317A2, WO02082904A2, WO0230410A2 , WO0220718A2, WO0210410A1, WO0183692A2, WO0 174163A1, WO0172329A1, WO0123001A2, WO01235 97A9, WO0057837A2, WO0055342A1, WO0028061A2 , WO9944645A1, WO9943360A1, WO9931982A1, WO9 915677A1, WO9914354A1, WO9915685A1, WO99100 13A1, WO9639530A3, WO9639416A1, WO9626286A1 and any of the rAAVs disclosed in WO9613598A2 (herein). All publications, patents, and patent applications referenced herein are incorporated by reference in their entirety. .

[0208] In certain embodiments, the rAAV in the pharmaceutical composition is RGX-121 (REGENXBI O Inc.), RGX-111(REGENXBIO Inc.), RGX-314( REGENXBIO Inc.), RGX-181(REGENXBIO Inc.), RGX-501 (REGENXBIO Inc.), Glybera (registered trademark) ipogene tiparvovec) (uniQure), Voretigene neparvovec(SPK-RPE65)(Spark Therapeutics ;MieraGTx UK II Ltd / Syne Qua Non Ltd / UCL ), rAAV2-CBSB-hRPE65(UPenn;NEI), rAAV2-hRP E65 (HMO), SPK-CHM (Spark Therapeutics), CNG A3-ACHM(AGTC), CNGB3-ACHM(AGTC), scAAV2-P1 ND4 (NEI), XLRS gene therapy drug (Biogen / AGTC), BMN-270 (Biomarin), SB-525 (Sangamo), DTX101 (Dimens) ion Therapeutics), SPK-9001 (SPK-FIX) (Spar k Therapeutics / Pfizer), AMT-060(uniQure / S t.Jude's Hospital), SB-FIX (Sangamo), scAAV 2 / 8-LP1-hFIXco(St.Jude's Hospital / UCL), A DVM-043 (Adverum), AVXS-101 (AveXis), rAAVrh 74.MCK.microdystrophin (NICHD), LGMD2D (NCH), rA AV1.CMV.human follistatin 344 (NCH), rAAVrh74.MHCK7 .DYSF.DV(NCH), ART-102(Arthrogen), intracerebral gene therapy Medicine (INSERM), CERE-110 (Ceregene), CERE-120 (Ce regene / Sangamo), AAV-hAADC(NIH), AAV2CUhCL N2 (Weill Cornell Medical College; Abeona Therapeutics), SAF- 301 (Lysogene), DTX301 (Dimension Therapeut ics), and TT-034 (Tacere Therapeutics) (Naso et al. BioDrugs. 2017;31(4): (See 317-334).

[0209] In certain embodiments, the rAAV in the pharmaceutical composition is AAV1, AAV2, AAV2tY F, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, and AAVrh10, AAV.rh20, AAV.rh39, AAV .Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV. Anc80L65, rAAV.7m8, AAV.PHP.B, AAV.PHP.eB, A AV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AA V.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HS C6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, one or more antigens selected from the group consisting of AAV.HSC15, or AAV.HSC16 In some embodiments, the pharmaceutical composition may include a component derived from a deno-associated virus serotype. The rAAV contains capsid proteins of the AAV8 or AAV9 serotype. In embodiments, the rAAV in the pharmaceutical composition comprises components derived from AAV9.

[0210] In certain embodiments, the pharmaceutical composition comprises a plurality of compounds. The product may be present in different hydrate forms, such as anhydrous, monohydrate, dihydrate, trihydrate, tetrahydrate, It consists of pentahydrate, hexahydrate, heptahydrate, octahydrate, nonahydrate, and decahydrate forms. The hydrate form is selected from the group including but not limited to:

[0211] In certain embodiments, the weight / volume concentration of the compound in the pharmaceutical composition may be any of the different hydrate forms. The molar equivalents of the compound may be expressed based on the anhydrous form of the compound. In some cases, the anhydrous form does not occur in nature.

[0212] In certain embodiments, the particular hydrate form of the compound in the pharmaceutical composition has an equivalent molar amount. These may represent different hydrate forms of the same compound.

[0213] In certain embodiments, the pharmaceutical composition comprises calcium chloride, e.g., calcium chloride in the dihydrate form. In other embodiments, the pharmaceutical composition does not include calcium chloride.

[0214] In certain embodiments, the pH of the pharmaceutical composition is about 7.4. The pH of the composition is about 6.0 to 8.8. In certain embodiments, the pH of the pharmaceutical composition is about 6. In certain embodiments, the pH of the pharmaceutical composition is about 6.0. In embodiments, the pH of the pharmaceutical composition is about 6.1. The pH is about 6.2. In certain embodiments, the pH of the pharmaceutical composition is about 6.3. In certain embodiments, the pH of the pharmaceutical composition is about 6.4. In certain embodiments, the pH of the pharmaceutical composition is about 6.6. In certain embodiments, the pH of the pharmaceutical composition is about 6.7. In certain embodiments, the pH of the pharmaceutical composition is about 6.9. In certain embodiments, the pH of the pharmaceutical composition is about 7.0. The pH of the composition is about 7.1. In certain embodiments, the pH of the pharmaceutical composition is about 7.2. In certain embodiments, the pH of the pharmaceutical composition is about 7.3. The pH of the composition is about 7.4. In certain embodiments, the pH of the pharmaceutical composition is about 7.5. In certain embodiments, the pH of the pharmaceutical composition is about 7.6. The pH of the pharmaceutical composition is about 7.7. In certain embodiments, the pH of the pharmaceutical composition is about 7.8. In certain embodiments, the pH of the pharmaceutical composition is about 7.9. The pH of the pharmaceutical composition is about 8.0. In certain embodiments, the pH of the pharmaceutical composition is about 8.1. In certain embodiments, the pH of the pharmaceutical composition is about 8.2. In certain embodiments, the pH of the pharmaceutical composition is about 8.3. In certain embodiments, the pH of the pharmaceutical composition is about 8.5. In certain embodiments, the pH of the pharmaceutical composition is about 8.6. In certain embodiments, the pH of the pharmaceutical composition is about 8.8. In certain embodiments, the pH of the pharmaceutical composition is about 8.9. It is 9.0.

[0215] In certain embodiments, the pH of the pharmaceutical composition is 7.4. In certain embodiments, the pH of the pharmaceutical composition is 6.0 to 8.8. 9.0. In certain embodiments, the pH of the pharmaceutical composition is 6.0. In certain embodiments, the pH of the pharmaceutical composition is 6.1. In certain embodiments, the pH of the pharmaceutical composition is 6.3. In certain embodiments, the pH of the pharmaceutical composition is 6.4. In certain embodiments, the pH of the pharmaceutical composition is 6.5. In certain embodiments, the pH of the pharmaceutical composition is 6.6. The pH of the pharmaceutical composition is 6.7. In certain embodiments, the pH of the pharmaceutical composition is 6.8. In certain embodiments, the pH of the pharmaceutical composition is 6.9. In certain embodiments, the pH of the pharmaceutical composition is 7.1. In certain embodiments, the pH of the pharmaceutical composition is 7.2. In certain embodiments, the pH of the pharmaceutical composition is 7.4. In embodiments, the pH of the pharmaceutical composition is 7.5. H is 7.6. In certain embodiments, the pH of the pharmaceutical composition is 7.7. In certain embodiments, the pH of the pharmaceutical composition is 7.8. 7.9. In certain embodiments, the pH of the pharmaceutical composition is 8.0. In certain embodiments, the pH of the pharmaceutical composition is 8.1. In certain embodiments, the pH of the pharmaceutical composition is 8.3. In certain embodiments, the pH of the pharmaceutical composition is 8.4. In certain embodiments, the pH of the pharmaceutical composition is 8.5. In certain embodiments, the pH of the pharmaceutical composition is 8.6. The pH of the pharmaceutical composition is 8.7. In certain embodiments, the pH of the pharmaceutical composition is 8.8. In certain embodiments, the pH of the pharmaceutical composition is 8.9. The pH of the product is 9.0.

[0216] In certain embodiments, the pharmaceutical compositions provided herein comprise a recombinant adeno-associated virus ( rAAV), as well as sodium chloride, magnesium chloride, potassium chloride, and dextrose A group consisting of poloxamer 188, sodium phosphate monobasic, and sodium phosphate dibasic In certain embodiments, the pharmaceutical composition comprises one or more compounds selected from calcium chloride. It further contains sodium.

[0217] In certain embodiments, the pharmaceutical compositions provided herein comprise a recombinant adeno-associated virus ( rAAV), as well as sodium chloride, magnesium chloride, potassium chloride, and dextrose A group consisting of poloxamer 188, sodium phosphate monobasic, and sodium phosphate dibasic In certain embodiments, the pharmaceutical composition comprises one compound selected from calcium chloride. It further includes

[0218] In certain embodiments, the pharmaceutical compositions provided herein comprise a recombinant adeno-associated virus ( rAAV), as well as sodium chloride, magnesium chloride, potassium chloride, and dextrose A group consisting of poloxamer 188, sodium phosphate monobasic, and sodium phosphate dibasic In certain embodiments, the pharmaceutical composition comprises two compounds selected from calcium chloride. It further includes

[0219] In certain embodiments, the pharmaceutical compositions provided herein comprise a recombinant adeno-associated virus ( rAAV), as well as sodium chloride, magnesium chloride, potassium chloride, and dextrose A group consisting of poloxamer 188, sodium phosphate monobasic, and sodium phosphate dibasic In certain embodiments, the pharmaceutical composition comprises three compounds selected from calcium chloride. It further includes

[0220] In certain embodiments, the pharmaceutical compositions provided herein comprise a recombinant adeno-associated virus ( rAAV), as well as sodium chloride, magnesium chloride, potassium chloride, and dextrose A group consisting of poloxamer 188, sodium phosphate monobasic, and sodium phosphate dibasic In certain embodiments, the pharmaceutical composition comprises four compounds selected from calcium chloride. It further includes

[0221] In certain embodiments, the pharmaceutical compositions provided herein comprise a recombinant adeno-associated virus ( rAAV), as well as sodium chloride, magnesium chloride, potassium chloride, and dextrose A group consisting of poloxamer 188, sodium phosphate monobasic, and sodium phosphate dibasic In certain embodiments, the pharmaceutical composition comprises five compounds selected from calcium chloride. It further includes

[0222] In certain embodiments, the pharmaceutical compositions provided herein comprise a recombinant adeno-associated virus ( rAAV), as well as sodium chloride, magnesium chloride, potassium chloride, and dextrose A group consisting of poloxamer 188, sodium phosphate monobasic, and sodium phosphate dibasic In certain embodiments, the pharmaceutical composition comprises six compounds selected from calcium chloride. It further includes

[0223] In certain embodiments, the pharmaceutical compositions provided herein comprise a recombinant adeno-associated virus ( rAAV), as well as sodium chloride, magnesium chloride, potassium chloride, and dextrose A group consisting of poloxamer 188, sodium phosphate monobasic, and sodium phosphate dibasic In certain embodiments, the pharmaceutical composition comprises all seven compounds selected from: It also contains calcium.

[0224] In certain embodiments, provided herein are pharmaceutical compositions comprising: (a) recombinant adeno-associated virus (rAAV); (b) sodium chloride, (c) magnesium chloride, (d) potassium chloride, (e) dextrose, (f) Poloxamer 188, (g) monobasic sodium phosphate, and (h) dibasic sodium phosphate; In this case, the recombinant adeno-associated virus (rAAV) contains the AAV capsid and its The vector genome includes a vector genome packaged in a portion, the vector genome including: ) AAV 5' inverted repeat (ITR) sequence; (ii) promoter; (iii) human TPP 1; and (iv) the AAV 3′ ITR.

[0225] In certain embodiments, the pharmaceutical composition further comprises calcium chloride.

[0226] In certain embodiments, the sodium chloride, the magnesium chloride, the potassium chloride , the dextrose, the poloxamer 188, the monobasic sodium phosphate, the dibasic sodium phosphate The sodium phosphate and calcium chloride may be present in anhydrous, monohydrate, dihydrate, Trihydrate, tetrahydrate, pentahydrate, hexahydrate, heptahydrate, octahydrate, nonahydrate, or 10 hydrate It is in the form of a hydrate.

[0227] In certain embodiments, the pharmaceutical composition comprises: (a) the rAAV; (b) sodium chloride at a concentration of about 8.77 g / L; (c) magnesium chloride hexahydrate at a concentration of about 0.244 g / L; (d) potassium chloride at a concentration of about 0.224 g / L; (e) calcium chloride dihydrate at a concentration of about 0.206 g / L; (f) anhydrous dextrose at a concentration of about 0.793 g / L; (g) poloxamer 188 at a concentration of about 0.001% (vol / vol); (h) monobasic sodium phosphate monohydrate at a concentration of about 0.0278 g / L, and (i) Sodium phosphate dibasic anhydrous at a concentration of about 0.114 g / L.

[0228] In certain embodiments, the vector genome concentration (VGC) of the pharmaceutical composition is about 1 x 10 11 GC / mL, approximately 3×10 11 GC / mL, approximately 6×10 11 GC / mL, approximately 1×10 12 G C / mL, approximately 3 × 10 12 GC / mL, approximately 6×10 12 GC / mL, approximately 1×10 13 GC / mL, approximately 2×10 13 GC / mL, approximately 3×10 13 GC / mL, approximately 4×10 13 GC / mL, approx. 5 x 10 13 GC / mL, approximately 6×10 13 GC / mL, approximately 7×10 13 GC / m L, about 8 x 10 13 GC / mL, approximately 9×10 13 GC / mL, or approximately 1 x 10 14 GC / mL, approximately 3×10 14 GC / mL, approximately 6×10 14 GC / mL, or approximately 1 x 10 15 In certain embodiments, the vector genome concentration (VGC) of the pharmaceutical composition is , 1×1011 GC / mL, 3 x 10 11 GC / mL, 6 x 10 11 GC / mL, 1x1 0 12 GC / mL, 3 x 10 12 GC / mL, 6 x 10 12 GC / mL, 1 x 10 13 G C / mL, 2 × 10 13 GC / mL, approximately 3×10 13 GC / mL, 4 x 10 13 GC / m L, 5 x 10 13 GC / mL, 6 x 10 13 GC / mL, 7 x 10 13 GC / mL, 8× 10 13 GC / mL, 9 x 10 13 GC / mL, or 1 x 10 14 GC / mL, 3 x 1 0 14 GC / mL, 6 x 10 14 GC / mL, or 1 x 10 15 GC / mL.

[0229] In certain embodiments, the pH of the pharmaceutical composition is in the range of about 6.0 to about 9.0. In embodiments, the pH of the pharmaceutical composition is about 7.4.

[0230] In certain embodiments, the rAAV in the pharmaceutical composition is the same recombinant rAAV as in the reference pharmaceutical composition. At least 2%, 5%, 7%, 10%, 12% %, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 10 0%, 2x, 3x, 5x, 10x, 100x, or 1000x stable. In some embodiments, the stability of the recombinant AAV may be assessed using one or more of the assays disclosed in Section IV and the Examples. Therefore, measure.

[0231] In certain embodiments, the stability of the rAAV in a pharmaceutical composition is measured by: . (a) rAAV infectivity, (b) rAAV aggregation levels, or (c) Levels of free DNA released by rAAV.

[0232] In certain embodiments, the pharmaceutical composition is a liquid composition. In certain embodiments, the pharmaceutical composition is a lyophilized composition or A reconstituted lyophilized composition.

[0233] In certain embodiments, the pharmaceutical composition is administered intracerebroventricularly (ICV), intracisternally (IC), intrathecally, Suitable for intracranial, intravenous, intravascular, intraarterial, intramuscular, intraocular, intramuscular, subcutaneous, or intradermal administration It has the following characteristics.

[0234] In certain embodiments, the coding sequence of (iii) of the rAAV in the pharmaceutical composition is The human CLN2 is an optimized human CLN2, which has at least one amino acid sequence similar to the native human coding sequence of SEQ ID NO:2. In certain embodiments, the rAAV in the pharmaceutical composition is at least 70% identical to (iii). The coding sequence is SEQ ID NO:3.

[0235] In certain embodiments, the rAAV capsid of the rAAV in the pharmaceutical composition is AAV9 or is its variant.

[0236] In certain embodiments, the promoter of the rAAV in the pharmaceutical composition is chicken β-actin In certain embodiments, the promoter of the rAAV in the pharmaceutical composition is a CBA promoter. The promoter contains the CBA promoter sequence and the cytomegalovirus enhancer element. It is a hybrid promoter.

[0237] In certain embodiments, the AAV 5'ITR and / or AA of the rAAV in the pharmaceutical composition The V 3'ITR is derived from AAV2.

[0238] In certain embodiments, the vector genome of the rAAV in the pharmaceutical composition further comprises polyA. In certain embodiments, the poly A is synthetic poly A or bovine growth hormone (BGH) GH), human growth hormone (hGH), SV40, rabbit β-globin (RGB), or is derived from modified RGB (mRGB).

[0239] In certain embodiments, the vector genome of the rAAV in the pharmaceutical composition further comprises an intron. In certain embodiments, the introns include CBA, human beta globin, IVS2, SV 40, bGH, alpha globulin, beta globulin, collagen, ovalbumin, or p5 3. It comes from

[0240] In certain embodiments, the vector genome of the rAAV in the pharmaceutical composition further comprises an enhancer. In certain embodiments, the enhancer includes a CMV enhancer, a RSV enhancer, or a Sir, APB enhancer, ABPS enhancer, αmic / bik enhancer, T TR enhancer, en34, and ApoE.

[0241] In certain embodiments, the rAAV vector genome in the pharmaceutical composition is about 3 kilograms in size. In certain embodiments, the base of the rAAV in the pharmaceutical composition is between about 5.5 kilobases. The ectodermal genome is approximately 4 kilobases in size.

[0242] In certain embodiments, suspension cell lines capable of producing rAAV are grown in suspension culture. The rAAV is produced in a pharmaceutical composition using a method comprising:

[0243] In another aspect, a method for treating a patient comprising administering to the patient a pharmaceutical composition provided herein. Provided herein are methods for treating CLN2 Batten disease in a patient. The pharmaceutical composition is administered in a therapeutically effective amount. In certain embodiments, the subject is a human.

[0244] In yet another aspect, provided herein is a kit comprising one or more containers and instructions for use. wherein one or more containers contain a pharmaceutical composition provided herein.

[0245] In certain embodiments, the pharmaceutical composition comprises 0.001% (weight / volume, 0.01 g / L) In certain embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.0005% at concentrations of 0.005g / L (weight / volume) to 0.05% (weight / volume, 0.5g / L) Poloxamer 188. In certain embodiments, the pharmaceutical composition contains 0.0001% (by weight) Poloxamer at concentrations of 0.001 g / L (weight / volume) to 0.01 g / L (weight / volume) In certain embodiments, the pharmaceutical composition comprises 0.0005% (weight / volume) Poloxamer at concentrations of 0.005 g / L) to 0.001% (weight / volume, 0.01 g / L) In certain embodiments, the pharmaceutical composition comprises 0.001% (weight / volume, 0 Poloxamer 188 at concentrations of 0.01g / L) to 0.05% (weight / volume, 0.5g / L) In certain embodiments, the pharmaceutical composition comprises 0.0005% (weight / volume, 0.005 In certain embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0. Contains poloxamer 188 at a concentration of 0.006% (weight / volume, 0.006 g / L). In this embodiment, the pharmaceutical composition has a concentration of 0.0007% (weight / volume, 0.007 g / L). In certain embodiments, the pharmaceutical composition contains poloxamer 188 at 0.0008% ( In certain embodiments, the composition contains poloxamer 188 at a concentration of 0.008 g / L (weight / volume). The pharmaceutical composition contains poloxamer at a concentration of 0.0009% (weight / volume, 0.009 g / L). In certain embodiments, the pharmaceutical composition comprises 0.001% (weight / volume, 0 In certain embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.01 g / L. , containing poloxamer 188 at a concentration of 0.002% (weight / volume, 0.02 g / L). In certain embodiments, the pharmaceutical composition has a concentration of 0.003% (weight / volume, 0.03 g / L). In certain embodiments, the pharmaceutical composition contains poloxamer 188 at 0.004% (by weight) In certain embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.04 g / L (by volume). The pharmaceutical composition contained poloxamer 188 at a concentration of 0.005% (weight / volume, 0.05 g / L). In certain embodiments, the pharmaceutical composition comprises 0.01% (weight / volume, 0.1 g / L) In certain embodiments, the pharmaceutical composition comprises poloxamer 188 at a concentration of 0.05% ( It contains poloxamer 188 at a concentration of 0.5 g / L (weight / volume).

[0246] As used herein, and unless otherwise specified, the term "about" means any amount within the meaning of any given This means within ±10% of the value or range.

[0247] The pharmaceutical compositions described herein can be administered by any suitable route or a combination of different routes. and is designed to deliver it to a subject in need thereof.

[0248] In yet another aspect, these nucleic acid sequences, vectors, expression cassettes and rAAV viruses The vectors are useful in pharmaceutical compositions, which are also pharmaceutically acceptable. Also includes carriers, excipients, buffers, diluents, surfactants, preservatives and / or adjuvants. Such pharmaceutical compositions may be used to infect such recombinant modified or artificial AAVs. Upon delivery, optimized TPP1 is expressed in the host cells.

[0249] These, including nucleic acid sequences, vectors, expression cassettes and rAAV viral vectors, For the preparation of a pharmaceutical composition, the sequence or vector or viral vector is preferably The sample is evaluated for contamination by conventional methods and then prepared into a pharmaceutical composition suitable for administration to a patient. Such formulations contain pharmaceutical additives to maintain pH at appropriate physiological levels. and / or a physiologically acceptable vehicle or carrier, such as buffered saline or Other buffers, e.g., HEPES, as well as, optionally, other drugs, pharmaceuticals, stabilizers, buffers, etc. This includes the use of buffers, carriers, adjuvants, diluents, surfactants, or excipients. In this case, the carrier is typically a liquid. Exemplary physiologically acceptable carriers include sterile paper. Examples include pyrogen-free water and sterile pyrogen-free phosphate buffered saline. A variety of such known carriers are described in U.S. Pat. Publication No. 7,413,422, which is incorporated herein by reference. In one embodiment, the carrier is an isotonic sodium chloride solution. In another embodiment, the carrier is a balanced salt solution. For long-term storage of the virus, the presence of glycerol or Tween 20 is recommended. The mixture may be frozen under reduced pressure.

[0250] In one exemplary specific embodiment, the carrier or excipient composition comprises 180 mM NaCl , 10 mM NaPi, pH 7.3, 0.0001% to 0.01% Pluronic The exact composition range of the saline component of the buffer solution is 160 ml. 180 mM NaCl. Alternatively, different pH buffers (potentially HEPES, sodium bicarbonate, TRIS) can be used. For this purpose, a buffer containing 0.9% NaCl is useful.

[0251] As used herein, the term "dose" refers to the total amount of a drug delivered to a subject over the course of treatment. or the amount delivered in a single unit (or multiple units or divided doses) The pharmaceutical viral composition may comprise a codon-optimized nucleic acid encoding TPP1 as described herein. Approximately 1.0 x 1 per dose is used to contain a quantity of replication-deficient virus carrying the sequence. 0 9 GC~approx. 1.0×10 16 GC range (including all integers or fractions within the range) In one embodiment, the composition contains at least 1×10 9 , 2 × 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , or 9×10 9 GC (including all integers or fractions in the range) In another embodiment, the composition is formulated to have at least 1 x 10 per administration. 10 , 2 × 10 10 , 3×1010 , 4×10 10 , 5×10 10 , 6×10 10 , 7× 10 10 , 8×10 10 , or 9×10 10 GC (all integers or fractions in a range) In another embodiment, the composition is formulated to contain at least Also 1×10 11 , 2 × 10 11 , 3×10 11 , 4×10 11 , 5×10 11 , 6×10 11 , 7×10 11 , 8×10 11 , or 9×10 11 GC(all integers in range In another embodiment, the composition is formulated to contain At least 1×10 12 , 2 × 10 12 , 3×10 12 , 4×10 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , or 9×10 12 GC(all in range In another embodiment, the composition is formulated to contain 100 mg of hydroxybenzoates (including any whole number or fraction thereof). , at least 1 x 10 per dose 13 , 2 × 10 13 , 3×10 13 , 4×10 13 , 5 x10 13 , 6×10 13 , 7×10 13 , 8×10 13 , or 9×10 13 GC(range In another embodiment, the compound is formulated to contain 0.05g of ... , the composition is administered in a dose of at least 1 x 10 14 , 2 × 10 14 , 3×10 14 , 4×1 0 14 , 5×10 14 , 6×10 14 , 7×10 14 , 8×10 14 , or 9×10 1 4 Formulated to contain GC (including any whole number or fraction within the range). In embodiments, the composition is administered at a dose of at least 1 x 10 15 , 2 × 10 15 , 3×10 1 5 , 4×10 15 , 5×10 15 , 6×10 15 , 7×10 15 , 8×10 15 ,or 9×10 15 Formulated to contain GC (including all integers or fractions within the range) In one embodiment, for administration to humans, the dose is 1 x 10 per administration. 10 ~Approx. 1×1 0 12 It can be a range of GCs (including all integers or fractions within the range). In embodiments, the composition is administered in a concentration of at least 7.5×10 12 GC(7.5×10 9 GC / g brain Mass)~2.7×10 15 GC(2.1×10 12 Formulated to contain (GC / g brain mass) The mass of an average human brain is approximately 1,300g to 1,400g. The compositions herein are known in the art. It is believed that this will be useful for children with brain masses in the range of 100-1500 mg / kg. All dosages are given, for example, by reference M. Lock et al., Hum Gene Ther Met. hods.2014 Apr;25(2):115-25. doi:10.1089 / qPCR or digital drop as described in hgtb.2013.131 by any known method, including that measured by double-delayed PCR (ddPCR). It may also be measured.

[0252] In one embodiment, an aqueous suspension suitable for administration to a Batten patient is provided. and a sexual suspension of approximately 7.5 × 10 per gram of brain, useful as a treatment for Batten disease. 9 GC or virus particles ~ approx. 2.1 x 10 12 The compositions described herein for GC or viral particles and recombinant adeno-associated virus (rAAV).

[0253] It may be desirable to administer multiple "booster" doses of the pharmaceutical compositions of the present invention. For example, at 6-month intervals or every 6 months after the first dose, depending on the duration of the transgene in the CNS. A booster dose may be delivered in a year. The fact that the antibody was not generated by the antibody should allow for additional booster doses.

[0254] Such booster doses and their necessity depend, for example, on TPP1 activity and the following performance Neurocognitive testing, as described in the examples, can be used by the attending physician to monitor. Such tests may be used to assess the status of the subject over time. Selection of the appropriate test depends on: Alternatively, the method of the present invention may also be used to treat single or multiple infections. A large amount of virus-containing solution was injected into the mice to measure TPP1 activity levels in normal subjects. This may include approaching acceptable levels.

[0255] These above doses may vary depending on the size of the patient, the viral titer used, the route of administration, and the method of administration. Approximately 100 microliters in various amounts of carrier, excipient, or buffer formulation depending on the desired effect. The dose may range from 100 mL to about 50 mL, inclusive of all values ​​within the range. In one embodiment, the volume is less than 10 mL. In one embodiment, the volume is about 750 μL. In one embodiment, the volume is about 1 mL. In another embodiment, the volume is about 2 mL. In one embodiment, the volume is about 3 mL. In another embodiment, the volume is about 4 mL. In one embodiment, the volume is about 5 mL. In another embodiment, the volume is about 6 mL. In one embodiment, the volume is about 7 mL. In another embodiment, the volume is about 8 mL. In one embodiment, the volume is about 9 mL. In another embodiment, the volume is about 10 mL. In one embodiment, the volume is about 11 mL. In another embodiment, the volume is about 12 mL. In another embodiment, the volume is about 13 mL. In another embodiment, the volume is about 14 mL. In another embodiment, the volume is about 15 mL. In another embodiment, the volume is about 16 mL. In another embodiment, the volume is about 1 L. In another embodiment, the volume is about 17 mL. In another embodiment, the volume is about 19 mL. In another embodiment, the volume is In another embodiment, the volume is about 21 mL. The volume is about 22 mL. In another embodiment, the volume is about 23 mL. In one embodiment, the volume is about 24 mL. In another embodiment, the volume is about 25 mL or greater. In this condition, the maximum injection volume is approximately 10% of the total volume of cerebrospinal fluid.

[0256] In one embodiment, the viral construct is administered to a small animal subject, such as a mouse, at a dose of about 100 micrograms. At least 1 x 10 mL in a volume of 0.5 mL to approximately 1 mL 9 ~Approx. 1×10 13 GC dose For larger veterinary subjects, the larger human doses and volumes mentioned above may be used. For a discussion of good practice for administering substances to various veterinary animals, see, for example, D iehl et al, J. Applied Toxicology, 21:15-23 (2001), which is incorporated herein by reference.

[0257] To reduce the risk of unwanted effects such as toxicity, the virus or other delivery vehicle It is desirable to utilize the lowest effective concentration of cyclohexyl methylpropional. Further doses within these ranges may be used for therapeutic purposes. The physical condition of the subject, preferably a human, to be treated, the age of the subject, and the degree of onset of the disorder The attending physician may make a selection based on this consideration.

[0258] Yet another aspect described herein is a method for treating Batten disease in a mammalian subject, In one embodiment, a method for delaying or stopping the flow of blood is preferably physiologically compatible. CLN2 native or modified forms suspended in a suitable carrier, diluent, excipient and / or adjuvant. The rAAV having the mutated or codon-optimized sequence can be administered to desired subjects, including human subjects, for therapeutic efficacy. This method may involve administering a nucleic acid sequence, an expression cassette, a rAAV genome, a plasmid, or the like. The method further comprises administering to the patient a vaccine, a vector, or a rAAV vector, or a composition containing the same, In one embodiment, the composition is delivered intrathecally. In another embodiment, the composition is delivered via ICV. In yet another embodiment, the composition is delivered into the tank using a dosage form suitable for treating Batten disease. The drug is delivered using a combination of routes of administration, including intravenous administration or other conventional routes of administration. It may be included.

[0259] For use in these methods, the volume and dosage of each dose, as further described herein, Viral titers are determined individually. Dosage, administration and regimen are determined according to the instructions in this specification. In another embodiment, the method comprises administering the composition in two or more doses (e.g., In another embodiment, the selected expression cassette (e.g., A second administration of rAAV containing a CLN2-containing cassette is administered at a later time point. Such time points may be weeks, months, or years after the initial administration. Such a second administration may, in one embodiment, be administered using an rAAV having a capsid different from that of the first administration. In another embodiment, the first and second administrations of rAAV are performed using the same rAAV. It has the same capsid.

[0260] In yet other embodiments, the compositions described herein may be administered as a single composition or as multiple compositions. Optionally, two or more different AAVs, or multiple viruses, may be delivered. (See, for example, WO2011 / 126808 and WO2013 / 049493) In another embodiment, the multiple viruses are different replication-deficient viruses (e.g., , AAV and adenovirus).

[0261] In accordance with the present invention, a "therapeutically effective amount" of hTPP1 is delivered as described herein to Achieve the desired result, i.e., treatment of Batten disease or one or more of its symptoms. The Unified Batten Disease Rating Scale (UBDRS), a comprehensive assessment system of performance, behavior, and abilities, is proposed. rarediseases.info.nih.gov / files / m Mink, J., The Unified Batten, available at ink.pdf See Disease Rating Scale. CLN2 Disease Clinical Rating Scale ( CRS) (i.e., developed by the University Medical Center Hamburg-Eppendorf, Germany) The Hamburg Scale (developed by This scale includes motor loss, seizure activity, vision loss, and speech loss. It incorporates assessment of disease progression in multiple functional areas: each functional area is compared to normal a score of "3" for a condition, a score of "2" for a mild or barely perceptible abnormality, and Severe abnormality is assigned a score of "1" and complete loss of function a score of 0. These scores are then summed to form a score, which is incorporated herein by reference. Steinfeld et al., Late infantile neurona l Ceroid lipofuscinosis: quantitative des cription of the clinical course in patie nts with CLN2 mutations,Am J Med Genet.2 002 Nov 1;112(4):347-54 total disability score was assigned to each patient. See also Wyrwich et al., An Ada pted Clinical Measurement Tool for the K ey Symptoms of CLN2 Disease,Journal of I nborn Errors of Metabolism & Screening,2 See also CLN2, Volume 6:1-7. N2 disease: Without treatment, motor and language function declined as measured using CRS. They experience a consistent and progressive loss of memory (Nickel et al., 2018). In CLN2, the goal of treatment is to limit disease progression. May be assessed by quantitative and qualitative assessment of symptoms using a scale or the UBDRS .

[0262] In another embodiment, the method further comprises performing additional tests, e.g., assays and neurocognitive tests. Such studies include those based on the UBDRS guidelines listed above. These tests include, but are not limited to: speech intelligibility, tongue protrusion, visual acuity, and Strength, tension (arms, legs, neck), strength (arms, legs), hand tapping, heel stomping, spontaneous movement Movement (ataxia), stereotypies, dystonia, myoclonus, tremor, chorea, dysmetria, gait disorder These include assessments of postural stability, seizures, behavior and mood, and overall health.

[0263] In one embodiment of the methods described herein, a single delivery of a composition described herein, e.g. For example, AAV delivery of an optimized CLN2 cassette may be effective in treating Batten disease in a subject. In another embodiment of the methods described herein, a single dose of the compositions described herein is used. Delivery, e.g., AAV delivery of an optimized CLN2 cassette, can be used to target CLN2-deficient individuals. It is useful in preventing Batten disease in elephants.

[0264] Thus, in one embodiment, the composition is administered before the onset of the disease. In another embodiment, the composition is administered prior to the onset of a neurological disorder. In one embodiment, neonatal treatment is administered within 8 hours of delivery, the first 1 Within 2 hours, the first 24 hours, or the first 48 hours, In another embodiment, the administration of a gene sequence, expression cassette, or vector In particular, for primates (human or non-human), the delivery of a newborn baby takes approximately 12 hours to approximately one week. Within a period of 2 weeks, 3 weeks, or about 1 month, or within about 24 hours to about 48 hours. In another embodiment, the composition is delivered after the onset of symptoms. In another embodiment, treatment is initiated after the first injection. Age after 1 year, or the first 2-3 years, age after 5 years, age after 11 years, or later In one embodiment, treatment begins at about 4 to about 12 years of age. In some embodiments, treatment begins after about age 4. In one embodiment, treatment begins after about age 5. In one embodiment, treatment begins after about age 6. In one embodiment, treatment begins after about age 7. In one embodiment, treatment begins at about age 8 or later. In one embodiment, treatment begins at about age 8 or later. Treatment begins after about age 9. In one embodiment, treatment begins after about age 10. In one embodiment, treatment begins after about age 11. In one embodiment, treatment begins after about age 12. However, treatment may be initiated at about 15 years of age, at about 20 years of age, at about 25 years of age, at about 30 years of age, at about 35 years of age, or at about 40 years of age. In one embodiment, in utero treatment can begin at or after about age 40. This is defined as administering to a fetus a composition described herein. David et al., Recombinant adeno-a associated virus-mediated in utero gene t ransfer gives therapeutic transgene expr ession in the sheep,Hum Gene Ther.2011 A pr;22(4):419-26. doi:10.1089 / hum.2010.00 7. See Epub 2011 Feb 2.

[0265] In another embodiment, the composition is re-administered at a later date. Optionally, multiple re-administrations are performed. Such re-administration can be performed using the same type of vector or a different viral vector. This may be achieved via viral delivery or via non-viral delivery as described herein.

[0266] The goals of the treatments described herein include limiting or halting the progression of Batten disease. Desirable outcomes of treatment include, but are not limited to, U Increase in either BDRS and / or CLN2 disease rating scale scores, TPP1 Increased activity or expression levels, increased motor function (or reduced progression of motor impairment) These include a decrease in cortical volume (or a decrease in the progression of cortical damage) and an increase in cortical volume (or a decrease in the progression of cortical damage) by MRI. Desired outcomes include reducing muscle weakness, increasing muscle strength and tone, or maintaining respiratory health. Other desirable endpoints include an increase in the level of tremors or seizures, or a reduction in tremors or seizures. The teacher can decide.

[0267] In yet another embodiment, any of the above methods are combined with another or secondary treatment. Second-line therapy is performed to identify these mutations or defects or their associated effects. Any currently known or unknown treatment that helps prevent, inhibit, or ameliorate any of the following: The secondary therapy may be administered before, simultaneously with, or after administration of the composition. In one embodiment, the secondary therapy includes administration of neurotrophic factors, antioxidants, and anti-apoptotic agents. These include non-specific approaches to maintaining retinal cell health, such as administration of retinal markers. Chi may be produced by the production of proteins, recombinant DNA, recombinant viral vectors, stem cells, fetal tissue, or This is achieved by injecting genetically modified cells. In one embodiment, the secondary therapy may include intracerebroventricular serpinase alfa (BMN 19 0). Schulz et al., Intrac erebroventricular cerliponase alfa(BMN 1 90)in children with CLN 2 disease:result s from a phase 1 / 2 open label,dose-escal ation study,J Inherit Metab Disease,39:S See 51. The recommended dose is 30–300 mg ICV infusion administered every other week. .

[0268] In one embodiment, the method for producing a recombinant rAAV comprises using a recombinant rAAV containing an AAV expression cassette as described above. The plasmid that encodes the AAV viral genome is then used to express the infectious AAV envelope or capsule. The plasmid is prepared in the presence of sufficient viral sequences to enable it to be packaged into capsids. Specific methods for producing rAAV vectors include culturing packaging cells containing the vector. The methods are described above, in the expression cassettes and genomes described above, and in the Examples below. In order to generate rAAV vectors capable of delivering codon-optimized CLN2 in May be used.

[0269] In certain embodiments of the present invention, the subject has Batten disease and is administered the components, compositions, and The methods are designed to treat. As used herein, the term "subject" refers to humans, veterinary or livestock animals, domestic animals or pets, and clinically relevant In one embodiment, these methods and The subject of the composition is a human. Other suitable subjects include, but are not limited to, mice, Examples of such animals include rats, dogs, cats, pigs, cows, sheep, and non-human primates. As used herein, the term "subject" is used interchangeably with "patient."

[0270] As used herein, the term "treatment" or "treating" refers to the treatment of Batten disease. one or more of the compounds or The term "treatment" is defined to encompass administering a composition to a subject. reducing the onset or progression of Batten disease, preventing the disease, or reducing the severity of disease symptoms in a given subject. Reducing the severity or slowing the progression of, including the progression of, neurological disorders; eliminating disease symptoms; and / or monitoring the progression of disease or the effectiveness of treatment. It can be seen.

[0271] In one embodiment, a coding sequence encoding a functional TPP1 protein is provided. "hTPP1" refers to the native TPP1 protein or its non-disease-associated native variants. at least about 50%, at least about 75%, or at least about 80% of the biological activity of the ant or polymorph At least about 80%, at least about 90%, or roughly equivalent, or more than 100% The term "TPP1" refers to a gene encoding the TPP1 protein.

[0272] Various assays are available to measure TPP1 expression and activity levels in vitro. See, for example, Example 2 below. The methods described herein also include batch It may be combined with any other therapy for treating marrow disease or its symptoms. Management of LN2 disease is complex. Due to the high symptom burden and rapid rate of functional decline, patients It requires extensive multidisciplinary medical care and extensive psychosocial support for families, but There are no management guidelines for this condition. Management strategies for CLN 2 disease, Pediatric Neurology 69(2017)10 See US Pat. No. 2,811,222. However, in certain embodiments, the standard of care is intraventricular therapy. Liponase alpha (BMN 190) may be included. lz et al,Intracerebroventricular cerlipo nase alfa(BMN 190)in children with CLN 2 disease:results from a phase 1 / 2 open l abel,dose-escalation study,J Inherit Met See Ab Disease, 39:S51. The recommended dose is 30 mg once every other week. ~300mg ICV infusion.

[0273] In certain embodiments, the AAV9.CLN2 vector is produced. Examples of suitable purification methods are described, for example, in US Pat. International Patent Application No. PCT / US2016 / 065970 and its priority documents, 2016 No. 62 / 322,071, filed April 13, and incorporated herein by reference. No. 62 / 226,357, filed December 11, 2015, entitled " “Scalable Purification Method for AAV9”) It is written.

[0274] For AAV viral vectors, quantification of genome copies (“GC”) contained in the formulation Any method known in the art can be used to measure the dosage of the present invention. The genome copy (GC) number of the replication-deficient viral composition of AAV can be measured. One method to perform a titration of GC numbers is as follows: 1. Measure purified AAV vector samples. First, it is treated with DNase to remove contaminating host DNA from the manufacturing process. The DNase-resistant particles are then heat-treated to release the genome from the capsid. The genome is then transfected with plasmids targeting specific regions of the viral genome (e.g., poly(A) signal). Quantify by real-time PCR using a timer / probe set. Another suitable method for measuring the number of qPCR or digital droplet PCR [Lock Martin, et al,Human Gene Therapy Methods.April 2014 ,25(2):115-125.doi:10.1089 / hgtb.2013.131 , published online before editing on December 13, 2013]. Alternatively, ViroCyt31 00 can be used for particle quantification or flow cytometry. SK McLaughlin et al., 1988, which is incorporated by reference in its entirety. 8 Optimized TPP1 code as described in J. Virol., 62:1963 An effective amount of recombinant adeno-associated virus carrying a nucleic acid sequence encoding the sequence is determined.

[0275] The replication-deficient virus composition is administered to a human patient in an amount of approximately 1.0 x 10 9 GC~approx. 9×10 1 5 GC (including all integers or fractions within the range), preferably 1.0 x 10 12 GC~ 2.7×10 15 The virus can be formulated into dosage units containing amounts of replication-deficient virus in the GC range. In one embodiment, the composition is At least 1 x 10 per dose 9 , 2 × 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , or 9×10 9 GC(all integers in range In another embodiment, the composition is formulated to contain At least 1×1010 , 2 × 10 10 , 3×10 10 , 4×10 10 , 5×10 10 , 6×10 10 , 7×10 10 , 8×10 10 , or 9×10 10 GC(all in range In another embodiment, the composition is formulated to contain 100 mg of hydroxybenzoates (including any whole number or fraction thereof). , at least 1 x 10 per dose 11 , 2 × 10 11 , 3×10 11 , 4×10 11 , 5 x10 11 , 6×10 11 , 7×10 11 , 8×10 11 , or 9×10 11 GC(range In another embodiment, the compound is formulated to contain 0.05g of ... , the composition is administered in a dose of at least 1 x 10 12 , 2 × 10 12 , 3×10 12 , 4×1 0 12 , 5×10 12 , 6×10 12 , 7×10 12 , 8×10 12 , or 9×10 1 2 Formulated to contain GC (including any whole number or fraction within the range). In embodiments, the composition is administered at a dose of at least 1 x 10 13 , 2 × 10 13 , 3×10 1 3 , 4×10 13 , 5×10 13 , 6×10 13 , 7×10 13 , 8×10 13 ,or 9×10 13Formulated to contain GC (including all integers or fractions within the range) In another embodiment, the composition is administered at a dose of at least 1 x 10 14 , 2 × 10 14 , 3×10 14 , 4×10 14 , 5×10 14 , 6×10 14 , 7×10 14 , 8×10 1 4 , or 9×10 14 GC (including all integers or fractions in the range) In another embodiment, the composition is formulated at a concentration of at least 1 x 10 per administration. 15 , 2× 10 15 , 3×10 15 , 4×10 15 , 5×10 15 , 6×10 15 , 7×10 15 , 8×10 15 , or 9×10 15 GC (including all integers or fractions in the range) In one embodiment, for administration to a human, the dose is formulated to contain 1 x10 10 ~Approx. 2.7×10 15 GC range (including all integers or fractions within the range) It can be said that:

[0276] In certain embodiments, the dose is about 1 x 10 9 GC / g brain mass ~ approx. 2.1×10 12 GC / g brain mass. In certain embodiments, the dose is about 3 x 10 10 GC / g brain mass ~ approx. 3 × 10 11 In certain embodiments, the range may be in the range of GC / g brain mass. The dose is approximately 5 x 10 10 GC / g brain mass ~ approx. 1.85×1011 GC / g brain mass range It may be within the range.

[0277] In one embodiment, the viral construct is administered in a volume of at least about 1 x 10 9 GC~approx. 2.1×10 1 5 , or approximately 1 × 10 11 ~5×10 13 These doses and The volume suitable for delivery of the desired concentration may be determined by one skilled in the art. For example, about 1 μL to 150 mL. A volume of about 100 ml may be selected, with larger volumes selected for adults. In one embodiment, the volume is about 1 Generally, for newborns, the appropriate volume is about 0.5 mL to about 10 mL. For older infants, about 0.5 mL to about 15 mL may be selected. For children, a maximum volume of about 30 mL may be selected. For children under 13 and teenagers, a maximum volume of approximately 50 mL may be selected. In yet other embodiments, the patient is administered a volume of about 5 mL to about 15 mL, or about 7.5 mL. A volume of up to about 10 mL may be selected for intrathecal administration. The patient can choose between a volume of approximately 5 mL to approximately 15 mL or a volume of approximately 7.5 mL to approximately 10 mL. Other suitable volumes and doses may be determined. The dose should be adjusted to balance the therapeutic effect against the side effects of the recombinant vector. The amount of the agent used may vary depending on the therapeutic application for which it is used.

[0278] The recombinant vectors described above may be delivered to host cells according to published methods. In certain embodiments, for administration to a human patient, saline, surfactant, and physiological saline are used. The rAAV is suitably suspended in an aqueous solution containing a salt or mixture of salts compatible with the To achieve this, the formulation is maintained at a physiologically acceptable pH, for example, pH 6 to 9, or pH 6.5 to 7.5. The pH of the cerebrospinal fluid is adjusted to a range of 7.0 to 7.7 or 7.2 to 7.8. The pH range is approximately 7.28 to 7.32, so for intrathecal or intracisternal delivery, a pH within this range is required. whereas for intravenous delivery, a pH of 6.8 to about 7.2 may be desirable. In one embodiment, the pH is about 7.3. However, other pH values ​​within the broadest range may be used. and subranges thereof may be selected for other delivery routes.

[0279] Select a suitable surfactant or surfactant combination from a range of non-toxic non-ionic surfactants. In one embodiment, for example, a polymer having a neutral pH and an average molecular weight of 840 Pluronic® F68 (B), also known as Poloxamer 188, A difunctional block copolymer surfactant terminated with primary hydroxyl groups, such as ASF Other surfactants and other poloxamers, i.e., polyoxyethylene (poly Two hydrophilic chains of polyoxypropylene (poly(propylene)) are adjacent to each other. SOL, a nonionic triblock copolymer consisting of a central hydrophobic chain of pyrene oxide UTOL HS 15 (Macrogol-15 Hydroxystearic Acid), LABRAS OL (Polyoxycaprylic Acid Glyceride), Polyoxy 10 Oleyl Ether, TWEE N (polyoxyethylene sorbitan fatty acid ester), ethanol and polyethylene glycol In one embodiment, the formulation contains a poloxamer. Polymers are commonly named with the letter "P" (for poloxamer) followed by a three-digit number. : The first two digits x 100 indicate the approximate molecular weight of the polyoxypropylene core, and the last number The x10 indicates the polyoxyethylene content. In one embodiment, poloxamer 188 is selected. The surfactant is present in an amount of about 0.0005% to about 0.001% of the suspension. It may be possible.

[0280] In one example, the formulation may contain, for example, sodium chloride, sodium bicarbonate, dextrose, Magnesium sulfate (e.g., magnesium sulfate 7H2O), potassium chloride, calcium chloride sodium phosphate dibasic, and mixtures thereof. Optionally, a buffered saline solution containing one or more of these in water may be included. For intracisternal or intravenous delivery, the osmolality should be within a range compatible with cerebrospinal fluid (e.g., approximately 275-300°C). approximately 290); see, for example, emedicine.medscape.com / arti See cle / 2093316-overview. In some cases, intrathecal or For intravessel delivery, the commercially available diluent may be used as a suspending agent, or a separate suspending agent and other optional It may be used in combination with an excipient, for example, Elliotts B® solvent. See Lukare Medical. In other embodiments, the formulation may contain one or more The above permeation enhancers may also be included. Examples of suitable permeation enhancers include, for example, mannitol. Sodium glycolate, sodium taurocholate, sodium deoxycholate , Sodium Salicylate, Sodium Caprylate, Sodium Caprate, Lauryl Sulfate sodium, polyoxyethylene-9-lauryl ether, or EDTA. do.

[0281] In another embodiment, the composition may comprise a carrier, solvent, stabilizer, diluent, excipient, and / or azido. Those skilled in the art will be able to determine the appropriate vaccine for the disease, taking into account the indication for which the introduced virus is intended. An appropriate carrier can be readily selected. For example, one suitable carrier is saline. It may also be formulated with various buffers (e.g., phosphate buffered saline). Exemplary carriers include sterile saline, lactose, sucrose, calcium phosphate, Examples of suitable buffers include cellulose, dextran, agar, pectin, peanut oil, sesame oil, and water. The liquid / carrier may be used to prevent rAAV from adhering to the infusion tubing, but may also prevent rAAV from adhering to the in vivo Components that do not interfere with AV binding activity should be included.

[0282] In one embodiment, the proposed composition of the AAV9.CB7.hCLN2 drug product is a 2 mL bottle. Freezing of the AAV9.CB7.hCLN2 vector in 1 mL of formulation buffer contained in a vial The proposed formulation buffer is 150 mM sodium chloride, 1.2 mM magnesium chloride. Calcium, 3mM potassium chloride, 1.4mM calcium chloride, 1mM sodium phosphate, 4.4 mM dextrose, and 0.001% poloxamer 188, pH 7.3. The proposed quantitative composition of the AAV9.CB7.hCLN2 drug product is shown in Table 1 below. Table 1. Suggested quantitative composition of AAV9.CB7.hCLN2 solution for injection, 1 mL / vial L [Table 1]

[0283] Optionally, the compositions of the invention contain, in addition to the rAAV and carrier(s), a preservative, or The formulation may contain other conventional pharmaceutical ingredients, such as chemical stabilizers. Suitable exemplary preservatives include chloramphenicol, ... Butanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, para Examples include benzophenone, ethyl vanillin, glycerin, phenol, and parachlorophenol. Suitable chemical stabilizers include gelatin and albumin.

[0284] The composition according to the invention may comprise a pharmaceutically acceptable carrier as defined above. Suitably, the compositions described herein are administered via injection, osmotic pump, intrathecal catheter, or the like. A pharmaceutical agent designed for delivery to a subject or for delivery by another device or route. an effective amount of one or more of A, suspended in a suitable carrier and / or mixed with a suitable excipient; In one example, the composition is formulated for intrathecal delivery. In one embodiment, the delivery route includes injection into the spinal canal, e.g., the subarachnoid space. In another embodiment, the delivery route is intrathecal (ICV) delivery. In yet another embodiment, the route of delivery is intracisternal (IC) injection (i.e., into the cisterna magna). (intrathecal delivery via image-guided suboccipital puncture).

[0285] The viral vectors described herein can deliver hTPP1 to a subject in need thereof (e.g., preparing a pharmaceutical agent for delivery to a subject (e.g., a human patient), providing functional TPP1 to the subject, and / or can be used to treat Batten disease. The course of treatment may optionally be repeated with the same viral vector ( For example, AAV9 vector) or different viral vectors (for example, AAV9 and AA Additionally, the viral vectors and non-viral vectors described herein may include repeated administration of the viral vectors. Other combinations using viral delivery systems may also be selected.

[0286] The hTPP1 cDNA sequences described herein can be cloned using techniques well known in the art. It can be produced in vitro and synthetically. For example, Xiong et al. ,PCR-based accurate synthesis of long DN A sequence,Nature Protocols 1,791-797(2 PCR-based precise synthesis of long DNA sequences (PAS) was performed as described in

[006] . ) method may be used. Dual asymmetric PCR and overlap extension PCR may be combined. The combined method is Young and Dong's Two-step total gene e synthesis method,Nucleic Acids Res.200 4;32(7):e59. Gordeeva et al., J Mic robiol Methods. Improved PCR-based gene synthesis method and its application to the Citrobacter freundii phytase gene co 2010 May;81(2):147-52. See also Epub 2010 Mar 10; also see Oligonucleotide Synthesis and and the following patents relating to gene synthesis, Gene Seq. 2012 Apr;6(1):1 See also US Patent No. 8,008,005; and US Patent No. 7,985,565. Further, for generating DNA via PCR, Kits and protocols for these are commercially available. These include Taq polymerase; OneT aq® (New England Biolabs); Q5® Takazhong Real-time DNA polymerase (New England Biolabs); and GoTaq Polymerases including, but not limited to, G2 Polymerase (Promega) DNA can also be prepared by transfection with a plasmid containing the hOTC sequence described herein. Kits and protocols are known and commercially available. It is commercially available as a QIAGEN plasmid kit; Chargeswitch® Pro Filter Plasmid Kit (Invitrogen); and GenElute ( Plasmid kits (Sigma Aldrich) are also available. Other techniques useful herein include those that eliminate the need for thermocycling. These methods typically use Bst D instead of heat. NA polymerase, Large Fragment (New England Biol A strand-displacing DNA polymerase, such as ATP, is used to separate the double-stranded DNA. A is produced by amplification using reverse transcriptase (RT), an RNA-dependent DNA polymerase. , may be generated from an RNA molecule. RT can then be generated by cleaving a DNA strand complementary to the original RNA template. The resulting DNA is called cDNA. This cDNA is then polymerized using PCR or other methods as described above. Further amplification can be performed by isothermal or RT-PCR. (registered trademark);GeneArt(registered trademark) (Life Technologies); and Integrated DNA Technologies, Custom DNA can also be produced commercially by companies not affiliated with the company.

[0287] The term "expression" is used herein in its broadest sense and refers to the expression of RNA or RNA. With respect to RNA, the terms "expression" or "translation" are used to specifically refer to the production of RNA and proteins. The expression is related to the production of peptides or proteins. It is also possible.

[0288] The term "translation" in the context of the present invention refers to the process by which an mRNA chain controls the assembly of an amino acid sequence into a protein. The term refers to the process in the ribosome by which proteins or peptides are produced.

[0289] It should be noted that the terms "a" or "an" refer to one or more. Thus, the term "a" (or "one or more," and "at least one" are used interchangeably herein. can be done.

[0290] The terms "comprise," "comprises," and "contain" "comprising" should be interpreted inclusively rather than exclusively. The terms "consist," "consisting," and variations thereof are used in The various embodiments herein may be interpreted as including, but not limited to, "including" or "comprising" any of the following: Although presented using the term "from" to "from," under other circumstances, related embodiments may also be used. "consisting of" or "consisting essentially of" and (essentially of) Let's say.

[0291] As used herein, "disease," "disorder," and "pathological condition" refer to an abnormal state in a subject. are used interchangeably to mean

[0292] As used herein, the terms "about" or "approximately" are used to specifically Unless otherwise stated, a variability of 10% from the given reference is implied.

[0293] As used herein, the term "modulation" or variations thereof refers to the modulation of one or more components of a biological pathway. refers to the ability of a composition to inhibit

[0294] Unless otherwise defined herein, technical and scientific terms used herein provides a general guide to those skilled in the art for many of the terms used by and in this application. "Terms and Conditions" have the same meaning as commonly understood by reference to publicly available documents.

[0295] III. Manufacturing method Further provided herein are methods for producing the rAAV described herein. In embodiments, the method comprises growing in suspension culture a suspension cell line capable of producing rAAV. This includes:

[0296] In certain embodiments, buffers may be used in the manufacturing methods. Exemplary buffers include: Examples include Tris, BisTris, BisTrisPropane, Phosphate, and HEPES. However, it is not limited to these.

[0297] In certain embodiments, the time range for the primary bioreactor process run of the suspension is The incubation period may be 1 to 10 days. In certain embodiments, the suspension is incubated in the primary bioreactor. The time range for carrying out the process may be 5 to 8 days. The length of time for the implementation of the primary bioreactor process in the liquid is 1 day, 2 days, 3 days, It may be 4, 5, 6, 7, 8, 9, or 10 days.

[0298] In certain embodiments, the time for carrying out the primary bioreactor process of the suspension Within the ranges, one or more of pH, dissolved oxygen, and temperature levels may be adjusted. In this form, within the time range for carrying out the main bioreactor process of the suspension, The pH level may be adjusted. In certain embodiments, the primary bioreactor process of the suspension The dissolved oxygen level may be adjusted during the time period for carrying out the process. In this form, within the time range for carrying out the main bioreactor process of the suspension, The temperature level may be adjusted.

[0299] In certain embodiments, transient transfection is performed after 1-10 days of cell growth. In certain embodiments, transient transfection may be performed on day 1, 2, or 3 of cell growth. The test may be performed on the 3rd, 4th, 5th, 6th, 7th, 8th, 9th, or 10th day after the test. In certain embodiments, cell growth is carried out in a culture medium containing DMEM and 10% FBS. Good too.

[0300] In certain embodiments, transient transfection is performed using pAAV.CB7.CI.CL N2.RBG.KanR vector genome plasmids, pAdDeltaF6, and pAA may be performed using a mixture containing the V29KanRGXRep2 AAV plasmid In certain embodiments, the mixture contains pAAV.CB7.CI in an amount of 0.1 to 100 mg. In certain embodiments, the vector genome plasmid CLN2.RBG.KanR is The mixture comprises pAdDeltaF6 in an amount of 1 to 500 mg. The product contains the pAAV29KanRGXRep2 AAV plasmid in amounts ranging from 1 to 500 mg. In certain embodiments, after transient transfection, the cells are incubated for 1 to 10 days. In certain embodiments, after transient transfection, the cells may be incubated for 1 day. Incubate for 2, 3, 4, 5, 6, 7, 8, 9, or 10 days. That's fine.

[0301] In certain embodiments, during vector recovery, the cell culture is treated with an amount of 0.1 to 10 mg of chloride. Magnesium may be supplemented. In certain embodiments, cell cultures may be supplemented with magnesium during vector recovery. , 0.1mg, 0.5mg, 1mg, 1.5mg, 2mg, 2.5mg, 3mg, 3.5 mg, 4mg, 4.5mg, 5mg, 5.5mg, 6mg, 6.5mg, 7mg, 7.5 Magnesium chloride in mg, 8mg, 8.5mg, 9mg, 9.5mg, or 10mg amounts It may be supplemented with

[0302] In certain embodiments, the rAAV purification process involves four steps: TFF Concentration and buffer exchange, affinity chromatography, ion exchange chromatography This may include filtration, as well as concentration and buffer exchange by TFF.

[0303] In certain embodiments, the tangential flow filtration concentrates and buffer exchange. The buffer solution may contain Tris, sodium chloride, and a pH ranging from 6.0 to 8.5. Typical buffers include Tris, Bis-Tris, Bis-Tris-Propane, Phosphate, and HEP. These include, but are not limited to, ES.

[0304] In certain embodiments, the buffer used for affinity chromatography is Tris , sodium chloride, and a pH in the range of 6.0 to 8.5. The buffer used for affinity chromatography is Tris, sodium citrate, and a pH in the range of 6.0 to 8.5. In certain embodiments, affinity chromatography may be used. The buffers used for chromatography were bis-tris propane, pluronic F68, and a pH in the range of 7 to 14. Exemplary buffers include Tris, Bis-Tris, Bis-Tris, These include, but are not limited to, trispropane, phosphate, and HEPES.

[0305] In certain embodiments, the purification process for rAAV includes ion exchange chromatography. In certain embodiments, the purification process for rAAV involves cation exchange chromatography. In certain embodiments, the purification process for rAAV comprises anion exchange chromatography. In certain embodiments, the buffer used for ion exchange chromatography comprises May contain bis-tris propane, pluronic F68, and a pH range of 4-14 Exemplary buffers include Tris, BisTris, BisTrisPropane, Phosphate, and H These include, but are not limited to, EPES.

[0306] IV. Assay IV-1. Assays Related to Methods for Treating Batten Disease Those skilled in the art will be able to identify and characterize the markers using the assays described herein and / or techniques known in the art (e.g., the compositions described herein using the assays described in WO2018209205A1 and testing methods, e.g., using the rAA provided herein in a method for treating Batten disease. V can be tested. Assay details are provided in Examples 1 and 2. Also, how to use such assays to test the rAAV provided herein. This section shows in more detail how it can be done.

[0307] Related assays: TPP1 in CLN2 Batten disease m1J in mouse models In vivo test, TPP1 m1J Natural history study of knockout mice, TPP1 m1J Knock Pharmacological studies in mice, assays to measure TPP1 enzyme activity, digital Intraventricular effects in mice using noninvasive full-time monitoring in the vivarium Evaluation of AAV9 delivery (ICV) in C57BL / 6 TPP1m1J knockout mice ), safety pharmacology assays, toxicity studies in mice, and Pharmacodynamic studies in rhesus monkeys and cynomolgus monkeys, vector biodistribution assays, and vector shedding assays These include, but are not limited to, assays, repeated dose studies, carcinogenicity studies, and other toxicity studies. It will not be done.

[0308] IV-2. Assays Related to Pharmaceutical Compositions Those skilled in the art will be able to identify and characterize the present invention using the assays described herein and / or techniques known in the art. , testing the compositions and methods described herein, e.g., testing the formulations provided herein. Assay details are provided in Examples 3 and 4. Examples 3 and 4 also How such assays can be used to test the formulations provided herein This shows in more detail how

[0309] Li et al.,2019 Cell & Gene Therapy Insi ghts, 5(4):537-547, which is incorporated by reference in its entirety. As noted above, exemplary assays include, but are not limited to: 1) Digital droplet PCR (ddPCR) for genome copy number measurement; (2) Analysis of AAV genome content and total capsid proportion by spectrophotometry; (3) Capsid Size exclusion chromatography for determining DNA distribution and purity; (4) Capillary Assessment of purity of capsid virus proteins using HPLC electrophoresis; (5) in vivo In vitro titration assay - for quantifying differences in infectivity of AAV vectors in vitro (6) relative infectivity as a reliable method; and (7) empty / total capsid ratio and size. Analytical ultracentrifugation (AUC) to measure the distribution of saturation.

[0310] A. Freeze / Thaw Cycle Assay A controlled freeze / thaw cycle can be performed in the freeze dryer. There is sufficient spacing on the shelf to accommodate four vials of buffer solution in a thermocouple. It can be done.

[0311] B. Temperature Stress Assay Temperature stress stability test was performed at 1.0 x 10 12 GC / mL at 37°C for 4 days These assays can be performed to assess the relative stability of the formulations provided herein.

[0312] Assays that can be used to assess stability include in vitro relative Titer (IVRP), vector genome concentration (VGC by ddPCR), and fluorescence These include, but are not limited to, DNA isolation, dynamic light scattering, appearance, and pH.

[0313] C. Long-Term Stability Assay Long-term development stability studies were conducted over a 12-month period to demonstrate the efficacy of -8 In vitro relative potency at 0°C (≦-60°C) and -20°C (-25°C to -15°C) and be able to demonstrate maintenance of other qualities.

[0314] D. In vitro relative potency (IVRP) assay Transducing HEK293 cells to relate ddPCR GC titers to gene expression and assaying cell culture supernatants for anti-VEGF Fab protein levels. HEK293 cells were cultured on three polysilicon plates and then in vitro bioassays were performed. -Plating overnight onto D-lysine-coated 96-well tissue culture plates Cells were then pre-infected with wild-type human Ad5 virus, followed by the Construct II reference standard. Transduce three independently prepared serial dilutions of the test substance, each preparation on a separate plate. Three days after transduction, the cell culture medium was removed from the plates. Collect and measure VEGF-binding Fab protein levels via ELISA. For A, block a VEGF-coated 96-well ELISA plate, It was then incubated with the collected cell culture medium to The anti-VEGF Fab was captured using a Fab-specific anti-human IgG antibody. Fab proteins captured with GF were detected. After washing, horseradish peroxidase ( Add HRP substrate solution, develop color, stop with stop buffer, and read on a plate reader. The absorbance or OD of the HRP product is plotted against the log dilution and each test The relative potencies of the substances were evaluated using a four-parameter logistic regression model after passing a parallel similarity test. For reference standards on the same plate fitted with a delta, the formula: EC50 reference ÷ EC The titer of the test substance is calculated from the weighted average of the three plates. The titer is reported as a percentage of the reference standard titer calculated.

[0315] To correlate ddPCR GC titers with functional gene expression, we used ddPCR in HEK293 cells. In vitro transfection and assay of the activity of the transgene (e.g., enzyme) Bioassays may be performed. HEK293 cells were cultured in triplicate in 96-well tissue culture plates. The cells are then plated overnight in a medium containing wild-type human adenovirus serotype 5. After pre-infection with the virus, three independently prepared serial dilutions of the enzyme reference standard and the test substance were The cells are transduced with a solution of 1000 μl ... Two days after transfection, the cells were lysed and treated with low pH to activate the enzyme and transfect the transgene (enzyme). Assay enzyme activity using a peptide substrate that increases fluorescent signal upon cleavage by RFU fluorescence was plotted against log dilution to determine the relative potency of each test substance in parallel parallel assays. The same plots were fitted with a four-parameter logistic regression model after passing the test. Calculate the EC50 for the reference standard using the formula: EC50 reference ÷ EC50 test substance The potency of the test substance was calculated as a percentage of the reference standard potency from the weighted average of the triplicate plates. and reported.

[0316] E. Vector Genome Concentration Assay ddPCR can also be used to assess vector genome concentration GC.

[0317] F. Free DNA analysis using a dye fluorescence assay SYBR® Gold nucleic acid gel stain that binds to DNA ("SYBR Gold") The fluorescence of the dye can be used to measure free DNA. Fluorescence can be measured using a fluorometer and quantified with DNA standards. Results in ng / μL are reported. It can be reported.

[0318] To convert the measured free DNA in ng / µL to a percentage of free DNA, two assays were performed. Total DNA can be estimated using two approaches. Total DNA in the samples was estimated using GC / mL (OD) measured by optical spectroscopy. (where M is the molecular weight of DNA and 1E6 is a unit conversion factor):

[0319] Estimated total DNA (ng / µL) = 1E6 × GC / mL (OD) × M (g / mol) / 6. 02E23

[0320] In the second approach, samples were incubated with 0.05% poloxamer 188 at 85°C for 20 min. can be heated and measured by SYBR Gold dye assay in heated samples The actual DNA collected can be used as the total amount. For example, the total D measured by SYBR Gold dye is assumed to be The NA measurements (compared to UV measurements) were 131% for the Construct II dPBS formulation and 131% for the Sucrose formulation. The Construct II modified dPBS containing the sucrose formulation was found to be 152% (free DNA Variations in the conversion of ng / µL to percentages can account for the range of reported results. For trend analysis, raw ng / µL can be used or a consistent method A measured ratio can be used.

[0321] G. Size Exclusion Chromatography (SEC) SEC was performed using Waters Acquity Arc instrument ID 0447 (C3PO). A Sepax SRT SEC-1000 peak column was used with a 25 mm path length flow cell. Ram (PN215950P-4630, SN:8A11982, LN:BT090, 5μ The mobile phase can be, for example, For example, 20 mM sodium phosphate, 300 mM NaCl, 0.005% poloxamer The column was heated to 88°C, pH 6.5, and the flow rate was 0.35 mL / min for 20 min. The data was collected at a sampling rate of 2 points / second and a resolution of 1.2 nm. A 25-point average smoothing at 214, 260, and 280 nm can be performed. The ideal target load may be 1.5E11GC. The sample contains approximately 1 / 2 of the ideal target. Either 50 μL, which is 1 / 3, can be injected, or 5 μL can be injected.

[0322] H. Dynamic Light Scattering (DLS) Assay Dynamic light scattering (DLS) was performed using a Corning 3540 384-well plate with a sample volume of 30 μL. This can be performed on a Wyatt DynaProIII using the rate per iteration. Ten acquisitions can be collected over 10 seconds each, with three replicate measurements per sample. The solvent used for the sample was determined, for example, Construct II in dPBS. "PBS" for construct II in modified dPBS containing sucrose sample, and "4% You can set the "sucrose" and data quality criteria (baseline, SOS, noise) Results that do not meet the criteria (e.g., fit) can be "marked" and excluded from the analysis. The low delay time cutoff for modified dPBS containing the sample was changed from 1.4 μs to 10 μs. , the sucrose excipient peak at approximately 1 nm results in an artificially low cumulant analytical diameter. It is possible to eliminate any influence that may result from this.

[0323] I. Differential Scanning Calorimetry Low-temperature differential scanning calorimetry (low-temperature DSC) was performed using a TA Instruments DSC25 Approximately 20 μL of sample was loaded into the Tzero pan. The sample can be crimped with a Tzero Hermetic lid. Equilibrate for 2 minutes, then cool to -60°C at 5°C / min, equilibrate for 2 minutes, then cool at 5°C / min Heat flow data can be collected in a conventional manner. .

[0324] J. Real-time buffer pH tracking INLAB COOLP can detect the pH of various formulation buffers down to -30°C The pH was monitored with a RO-ISM low-temperature pH probe. The pH probe was then immersed in the buffer solution. Use a film to seal the gap between the Falcon tube and the pH probe to prevent contamination and evaporation. The probe was placed in a -20AD freezer along with the Falcon tube. The pH and temperature were maintained for approximately 20 hours or until the pH vs. temperature behavior achieved a repeatable pattern. The temperature change caused by the automatic defrosting process was recorded every 2.5 minutes. Stress conditions for the pH stability of the buffer solution were generated.

[0325] K. Osmolality Osmometers measure osmotic pressure using the technique of freezing point depression. The NI was 50mOsm / kg, 850mOsm / kg, and 2000mOsm / kg. This can be performed using a ST traceable standard. The fluid can be used to determine the system suitability of the osmometer.

[0326] L. Density measurement Density is measured using an Anton Paar DMA500 densitometer against water. Between samples, the densitometer can be washed with water, then methanol, and air-dried. . M. Viscosity measurement Viscosity can be measured by methods known in the art, such as the United States Pharmacopoeia (US Pharmacopoeia), published in 2019. P) and earlier versions, which are incorporated herein by reference in their entireties. It can be measured using methods known in the art.

[0327] N. Viral Infectivity Assay Francois,et al. Molecular Therapy Method ds & Clinical Development(2018)Vol.10,pp 223-236 (incorporated herein by reference in its entirety). Sea urchin TCID 50 Infectious titer assays can be used. Using the relative infectivity assay described in filed provisional application 62 / 745,859, This can be done.

[0328] O. Crystallization and glass transition temperatures An exemplary method is described in Croyle et al., 2001, Gene Ther. 8( 17):1281-90, which is incorporated herein by reference in its entirety. ).

[0329] IV-3. Assays and Production Methods Related to Recombinant Adeno-Associated Virus (rAAV) Those skilled in the art will be able to identify and characterize the markers using the assays described herein and / or techniques known in the art (e.g., the compositions described herein using the assays described in WO2018209205A1 and methods may be tested, for example, the rAAV provided herein. Details are provided in Example 5. Example 5 also describes how to use such an assay. The present disclosure provides more details on how the rAAV provided herein can be tested using .

[0330] The bulk drug substance manufacturing process is summarized in the flow diagrams shown in Figures 38 and 39. The bulk drug substance was cultured in polyethylene glycol in HEK293 cells using the three plasmids described in Example 5. It can be produced by polyethylenimine (PEI)-mediated transient transfection.

[0331] Relevant assays may include, but are not limited to: transient transfection transfection, vector recovery, vector purification processes, e.g., tangential fractionation Concentration and buffer exchange by low filtration and affinity chromatography, ion Exchange chromatography and optional concentration or dilution by TFF Say. [Example]

[0332] The following examples are for illustrative purposes only and are not intended to limit the invention. It's not that.

[0333] Example 1: AAV9.CB7.hCLN2 is a model of CLN2 Batten disease, TP P1 m1J Improve survival and neuropathology in mice This example describes the use of TPP1, a model of CLN2 disease. m1J Using knockout mice To evaluate the rAAV provided herein, e.g., AAV9.CB7.hCLN2, This invention provides a method that can be used to

[0334] TPP1 m1J Knockout mice, as well as alternative mouse models of CLN2, are useful for detecting and treating diseases in humans. This shows the characteristics of CLN2 disease in the 2000s (Sleat et al., 2004. J. Neurol. osci;24(41):9117-26.;Geraets et al,2017. PLoS One;12(5):e0176526). The purpose of this study is to Through assessment of lifespan, TPP1 activity, and pathology in the brains of mice lacking AAV9.CB 7. To establish the efficacy of hCLN2.

[0335] method

[0336] TPP1 m1J Groups of knockout mice (N=10 / sex / group; approximately 4 weeks old) were injected with AAV 9. Single intracerebroventricular (ICV) injection (5 μL) of CB7.hCLN2 was administered at 0, 1.25 × 10 10 , 5×1010, 2×10 11 or 8.5 x 10 11 GC / animal dose After 9 weeks of treatment, animals were either euthanized (5 animals / sex / group) or continued on the study ( The effect of AAV9.CB7.hCLN2 on lifespan was assessed in mice (5 mice / sex / group). TPP1 m1J The knockout mice are genotyped.

[0337] The following endpoints were assessed: in-life observation, TPP1 activity (serum, brain [right hemisphere]) , spinal cord [thoracic] and liver), anti-TPP1 antibodies [serum] and neuropathology. The results showed neuronal loss in the brain, cervical spine, and lumbar spinal cord, as well as intralysosomal accumulation of autofluorescent storage materials. , astrocytosis (GFAP) and microglial activation (CD68) were assessed.

[0338] Enzyme assay to measure the cleavage of the non-fluorescent AAF-AMC substrate by TPP1 into non-fluorescent AMC. The enzymatic activity of TPP1 was assessed using a simple assay. The presence of anti-TPP1 antibodies was assessed by a solution bridging immunoassay.

[0339] result

[0340] As shown in Figure 2, AAV9.CB7.hCLN2-treated TPP1 m1J Knockout AAV9.CB7.hCLN2 increased the survival rate of TPP1-deficient mice. ≥ 2 × 10 11 The results showed that administration of GC / animal at a dose of 100 mg / kg / day improved survival. The highest dose showed 100% survival in both females.

[0341] Unprocessed TPP1 m1J Clinical signs observed in knockout mice gradually disappeared after 8 weeks. Signs commonly observed include tremors and splayed hind legs commonly associated with this disease model. In surviving AAV9.CB7.hCLN2-treated animals, clinical signs were observed within 8 days. The signs were similar to those observed in untreated animals from week 1 onwards (tremors and abnormal gait), but the food Intake was normal and the animals continued to gain weight.

[0342] As shown in Figure 3, TPP1 activity was significantly increased by AAV9.CB7.hCLN2-treated TPP1 m1J TPP1 activity was increased in the brains of all AAV9.CB7 knockout mice. TPP1 activity was increased in hCLN2-treated animals, with no sex-related differences. TPP1 activity was ≥ 5 × 10 10 The GC / animal ratio was comparable.

[0343] As shown in Figure 4, TPP1 activity was significantly increased by AAV9.CB7.hCLN2-treated TPP1 m1J Increased in the spinal cord of knockout mice treated with AAV9.CB7.hCLN2 A dose-related increase in TPP1 activity was observed in treated animals.

[0344] At week 9, a high incidence of anti-TPP1 antibodies was observed, with the lowest signal observed at the higher doses. This could be due to assay interference or reflect the induction of immune tolerance. be.

[0345] As shown in Figure 5, astrocytosis was significantly reduced in AAV9.CB7.hCLN2-treated mice. TPP1 m1J AAV9.CB7.hCLN2 was found to be involved in the thalamus. Astrocytosis (GFAP) in the VPM / VPL and S1BF cortex The animals at the highest dose were comparable to WT animals.

[0346] As shown in Figure 6, microglial activation was significantly enhanced in AAV9.CB7.hCLN2-treated T cells. PP1 m1JAAV9.CB7.hCLN2 was found to be involved in the thalamus ( Reduced microglial activation (CD68) in the VPM / VPL and cortex (S1BF) ≧2×10 11 In GC / / animals, CD68 immunoreactivity was comparable to that in WT animals. there were.

[0347] As shown in Figure 7, AAV9.CB7.hCLN2-treated TPP1 m1J Knockout AAV9.CB7.CLN2 increased hepatic TPP1 activity in mice. Increased TPP1 activity in rats, with a greater increase observed in males.

[0348] As shown in Figure 8, AAV9.CB7.hCLN2-treated TPP1m1J knockout AAV9.CB7.hCLN2 increased serum TPP1 activity in mice. It increased TPP1 activity in the supernatant, with a greater increase observed in males.

[0349] conclusion

[0350] AAV9.CB7.hCLN2 in a biologically relevant animal model of CLN2 disease A single dose of α-glucan increased TPP1 activity in the brain, spinal cord, and liver, and actin activity in the thalamus and cortex. Improved survival was demonstrated through reduced eosinophilic cytosis and microglial activation.

[0351] Increasing TPP1 activity in the brain alone did not improve survival rates, so this model demonstrated the potential therapeutic benefits of transducing the spinal cord and liver.

[0352] Gender-related differences in liver transduction efficiency may be due to serotype, promoter, or transgene. Independent of offspring, rodent-specific androgen-dependent effects on AAV uptake and (Lonning et al., 2002. Molecular Ther apy Vol.5,No.5;Davidoff et al,2003. Bloo d.15;102(2):480-8). The minimum effective dose is 2 × 10 11 GC / Animal It is possible.

[0353] Example 2: Studies on AAV9.CB7.hCLN2 This example demonstrates the use of rAAV provided herein, e.g., AAV9.CB7.hCLN2. Provides methods that can be used to evaluate efficacy in non-clinical studies and in humans. This includes testing.

[0354] I. Non-clinical studies A. Nonclinical Pharmacology A series of in vivo preclinical pharmacology studies were performed with AAV9.CB7.hCLN2. These studies were performed in a relevant mouse model of CLN2 disease, demonstrating AAV delivery to the CSF. 9. Improved survival and brain TPP1 activity after intracerebroventricular (ICV) administration of CB7.hCLN2. Neuropathological analysis showed increased neuronal loss in the brain, cervical spine, and lumbar spinal cord. The decrease in the number of fluorescent proteins, as well as the intralysosomal accumulation of autofluorescent storage materials, astrocytosis, and microenvironmental degradation Logarithmic activation was demonstrated.

[0355] In vivo pharmacological studies were performed using TPP1 m1J Knockout (KO) mice, delayed infancy Biologically relevant features of endothelial neuronal ceroid lipofuscinosis (LINCL)-Batten disease (CLN2) The study was carried out using appropriate disease models. m1J Knockout mice lack the CLN2 gene The gene has a single nucleotide mutation in the splice donor site downstream of exon 8, which causes soluble lysosomal Mutations in this gene encode the endothelial enzyme TPP1, which causes lipofuscitation, most notably in neural tissue. Lipofuscin accumulation leads to inflammation, glial cell activation, and This correlated with subsequent neuronal degeneration. Neuronal degeneration affects the brain, spinal cord, and motor nerves. TPP1 m1J Natural history studies using knockout mice have shown that Mice with this condition exhibit early onset of clinical symptoms, rapid progression of abnormal phenotypes, and shortened lifespan. , and CLN in humans, including similar pathophysiological, biochemical, and functional changes. The characteristics of the two diseases were shown. m1J The changes observed in the knockout mice were TPP1, an alternative mouse model of 2 tm1Plob or Cln2 R207X / R20 7X These changes were similar to those observed in mice (Sleat DE, et al. Am J Hum Genet.1999;64(6):1511-23;Geraets e t al,2017.PLoS One;12(5):e0176526) Therefore ,Overall, TPP1 m1J Knockout mice provide a biologically relevant model of CLN2 disease It is considered a rodent model.

[0356] All mouse studies were performed using CSF administration, as intracisternal administration is not possible in young mice. The AAV9.CB7.hCLN2 was injected intravenously into the vein of the vein. 9 The biodistribution of vector-based products is comparable to that of intrathecal injection into the cisterna magna (the proposed clinical route of administration). Preclinical studies in multiple species have demonstrated the efficacy of AAV in the distribution of AAV in the CNS and peripheral tissues. It has been shown that the quality of the fabric is equivalent between these routes of administration (Haurigot V, et al. J Clin Invest.2013;123(8):3254-71;H inderer et al,2017;McLean et al,2014,Bel le et al., 2019). Therefore, this is a promising strategy for AAV9.CB7 in mice. .It is an acceptable approach to evaluate the efficacy and safety of hCLN2.

[0357] A summary of the studies is shown in Table 2. Table 2. Non-clinical studies using AAV9.CB7.hCLN2 [Table 2] GC: genome copy; GLP: Good Laboratory Practice; ICV: intracerebroventricular; CM: intrathecal cisterna magna; I TL: intrathecal lumbar; KO: knockout; NA: not applicable; MFD: maximum feasible dose Amount; ROA: route of administration; TPP1: tripeptidyl peptidase-1.

[0358] 1. In vivo pharmacology a) TPP1 m1J Natural history study of knockout mice The purpose of this study is to m1J Phenotype and histopathology of knockout mice, and human The objective of this study was to characterize the ability of an animal model to recapitulate key features of CLN2 disease in humans. This natural history study included 5 groups of 20 animals (10 animals / sex / group) for a maximum of 2 Groups of animals were euthanized at 4 and 14 weeks of age to assess disease progression. The median survival time was 16 weeks for male knockout animals and 10 weeks for female knockout animals. 19.5 weeks, which is comparable to other mouse models of CLN2 disease (Sleat D E,et al. J Neurosci,2004;24(41):9117-26. ;Geraets et al,2017.PLoS One;12(5):e0176 526). TPP1 m1J Knockout mice showed a decrease in the cerebrum and The liver showed a lack of TPP1 enzyme activity (undetectable levels). Phenotypic onset was approximately 3 months of age based on gait abnormalities. The most prominent disease symptom was Seizures were observed earlier in males than in females, and sudden death occurred at 3.5 months of age after seizures. TPP1 was associated with increased stress due to conflicts with peers within the group. m1J Knocka From one week after birth (and possibly even before birth), the lack of TPP1 enzyme activity in the oocytes Lipofuscin granules, also known as lysosomal digested lipid-containing granules, are present in the cytoplasm of neurons. Accumulation of lipofuscin in the cytoplasm of neurons occurred in 1-month-old T PP1 m1J Hematoxylin and eosin (H&E) staining of knockout animals revealed It became clear that astrocyte activation or astrocytosis (an indicator of neuroinflammation) This correlated with an increase in TPP1. m1J Knockout mice show progressive loss of motor function Patients experienced worsening of the condition, gait abnormalities, tremors, seizures, weight loss and inability to eat, and early death. Neuroinflammation, identified by GFAP staining, peaked at 3 months of age and was associated with TPP1 m1J The TPP1 knockout animals were found to have a high risk of developing the disease. m1J Knocka The animals showed a progressive accumulation of storage material in neurons of the CNS.

[0359] These results suggest that TPP1m1J Knockout mice show early onset of clinical symptoms , rapid progression of abnormal phenotypes and shortened life span, as well as similar pathophysiological and biochemical These data show the hallmarks of CLN2 disease in humans, including phenotype, phenotype, and functional changes. Based on the data, TPP1 m1J Knockout mice were generated using the AAV9.CB7.hCLN2 vector. It was considered an appropriate model for assessing efficacy.

[0360] b) TPP1 m1J Pharmacology of AAV9.CB7.hCLN2 in knockout mice studies TPP1 m1J Therapeutic efficacy of AAV9.CB7.hCLN2 in knockout mice To test this, AAV9.CB7.hCLN2 was transfected into 1-month-old C57Bl / 6 TPP 1 m1J Knockout mice (10 mice / sex / group) were treated with 0 (PBS), 3 × 10 9 GC / Animals, or 3x10 11 The dose was 100 mg / animal per day and administered ICV. Floor findings, motor coordination (rocking rotarod test), learning ability (learning rotarod test) ), body weight, TPP1 activity, and anti-TPP1 antibodies. Twenty-six weeks after injection, in surviving animals, TPP1 activity in the brain and liver, and intrahepatic levels of vector The distribution and brain histopathology modifications associated with AAV9.CB7.hCLN2 treatment were evaluated. There were no adverse clinical findings. A dose-dependent increase in TPP1 levels was observed, which was associated with improvements in both inflammatory bowel disease and inflammatory bowel disease. 11 GC / animal, TPP1 m1J The survival rate of the knockout mice was 26 weeks after , 100% in males and 70% in females (Figure 9).

[0361] At 11 weeks after administration, motor coordination (latency to fall from a rocking rotarod) ) is 3 x 10 11 TPP1 in GC / animals only m1J Knockout mice equivalent to WT animals However, at 20 weeks post-treatment, there was no significant difference in the survival rate at this dose compared to WT controls. The latency to fall was reduced. In contrast, the latency to fall was reduced at both 13 and 20 weeks after administration. In motor learning, 3 × 10 11 WT mice and TPP1 in GC / animal m1J With the mouse There was no difference between 3 × 10 9 GC / TPP1 in animals m1J Luck with knockout mice No improvement in dynamic coordination or learning, untreated TPP1 m1J It was equivalent to that of a knockout mouse. TPP1 m1J In knockout mice, lysosomal cytoplasmic changes in neurons throughout the brain were observed. Accumulation of TPP1 in the untreated TPP1 m1J Compared to knockout mice , 3×10 11 GC / TPP1 in animals m1J In knockout mice, lysosomal storage The amount of stored substances was reduced, and the morphology of neurons was considered normal. Assessment of the brain for cytotoxicity (cytotoxicity staining) demonstrated that AAV9.CB7.hCLN2 inhibited cytotoxicity in the cortex and hippocampus. It was shown that astrocytosis could be prevented with 100% efficiency (Figure 1 0). A decrease in astrocytosis was observed in the brainstem (Fig. 10). 9 GC / Dynamic There was no effect on survival or pathology. Collectively, these results support the Therapeutic efficacy of AAV9.CB7.hCLN2 to prevent clinical symptoms and histopathology of patients was demonstrated.

[0362] TPP1 enzyme activity was measured in serum samples collected 10 and 26 weeks after injection. 3×10 11 In GC / animals, at 10 weeks post-injection, animals had supraphysiological TPP1 serum activity. At week 10, all AAV9.C B7.hCLN2-treated animals, especially those treated at low doses, showed high levels of anti-T cells in the serum. The high-dose treated males developed PP1 antibodies, possibly due to partial tolerance or high T A milder humoral immune response to the transgene due to interference with the assay by PP1 It seemed that the following symptoms were expressed:

[0363] At the study endpoint, TPP1 enzyme activity was measured in three organs: the cerebrum, the cerebellum / brainstem, and the liver. In the liver, PBS-treated WT mice and high-dose TPP1 m1J The results were compared between mice. For tissues, TPP1 activity was measured by the treatment with TPP1. m1J In knockout mice Although the expression level was high, this indicates the efficacy of AAV9.CB7.hCLN for restoring TPP1 function. 2 ICV therapy has shown high efficacy. m1J Except for knockout mice, No sex differences were observed within the groups. At the high dose, hepatic TPP1 activity was significantly higher in males than in females. was 10 times higher.

[0364] In this study, the minimum effective dose was 3 × 10 11 GC / animal, which means survival The rate, in-life observation, reduction of lysosomal storage material, normal neuronal morphology, and astrocytosis Based on prevention of idiopathic encephalopathy, 7.5 × 10 11 Corresponding to GC / g brain (estimated brain weight 0.4g) At this dose, the mean TPP1 activity in the brain (cerebrum and cerebellum combined) was 19205kJ / kg. U / mg / hour in the liver, 99991 U / mg / hour in the serum, and 177 U / μL / hour in the serum. there were.

[0365] c) Using non-invasive full-time monitoring with Digital Vivarium Evaluation of the efficacy of intracerebroventricular AAV9.CB7.hCLN2 in mice TPP1 m1J Efficacy of AAV9.CB7.hCLN2 in knockout mice To test, a digital vivarium (Vium, Inc., USA) Using unbiased, non-invasive, full-time monitoring, AAV9.CB7.hCL N2, 3 x 10 11 GC / animal, 7-week-old TPP1 m1J Knockout mice (7 The treated animals were administered ICV to age-matched vehicle-treated rats (1 male and 6 female). TPP1 m1J Knockout mice (5 males and 5 females) and WT mice (5 males The following endpoints were recorded continuously, including overnight These included activity, diurnal activity, respiratory rate, and circadian rhythm. Recording began 2 weeks after LN2 ICV injection (9 weeks of age) and was continued until scheduled sacrifice (1 week after injection). The animals were then followed for 6 weeks, 23 weeks of age, or until unscheduled death (presumably at age 14 or 15 weeks) or earlier. Brain and liver weights and evaluation of brain and liver histopathology were performed in surviving animals. Processing TPP1 m1J Knockout mice exhibit tremors, hunched posture, abnormal gait, and seizures. The animals were either euthanized due to worsening condition or found dead. Clinical signs were observed at 14.9 weeks. On the other hand, AAV9.CB7.hCLN2-treated TPP1 m1J Knocka 54% of the mice (7 of 13) were clinically unremarkable throughout the study. The mean age at first clinical event was 1.5 years. m1J Knockout vehicle-treated mice At 16.1 weeks, six AAV9.CB7.HCLN2-treated TPP1 mice were m1J Knockout Ma The age of the mice was 18.6 weeks. Seven AAV9.CB7.hCLN2-treated TPP1 mice m1J of The checked-out mice showed no clinical signs.

[0366] AAV9.CB7.hCLN2: 3 x 10 11 Well tolerated at GC / animal doses TPP1 m1J Vehicle-treated TPP1 extended the lifespan of knockout mice. m1J Knock The median survival time of the euthanized mice was 17.6 weeks for males and 17.4 weeks for females. On average, death occurred 1.5 weeks after the onset of clinically visible signs. At the endpoint (16 weeks post-injection, 23 weeks of age), 57% (4 of 7) of treated males 67% (4 of 6) of the females treated with TPP1 survived, whereas all untreated TPP1 m1J Knockout mice died by 19 weeks of age (Fig. 11). AAV9.CB7. hCLN2-treated TPP1 m1J The knockout mice were compared with the WT mice throughout the study. The vehicle-treated mice maintained their body weight close to that of the control group, whereas the vehicle-treated mice gradually lost weight from 13 weeks of age. Respiration rates were similar in all groups, and no effect of genotype or treatment could be detected. Normal healthy mice are more active during the dark period at night and less active during the light period during the day. Both male and female vehicle-treated TPP1 mice showed a distinct biphasic circadian locomotor pattern. m1 J Knockout mice begin to lose their biphasic characteristics and show reduced activity during the dark phase around 16 weeks of age. AAV9.CB7.hCLN2-treated mice were treated identically to WT mice until the end of the study. Vehicle-treated TPP1 showed circadian motility characteristics. m1J The knockout mice were WT The nighttime movement speed was reduced compared to that of the control mice, and by 7.7 weeks after injection (14.7 weeks of age), statistical Conversely, a significant decrease in TPP1 expression was observed in AAV9.CB7.hCLN2-treated mice. m1J Knockout mice maintained their nighttime activity levels, which was consistent with vehicle-treated TPP. 1 m1J Statistically significantly higher than in knockout mice, and significantly higher than in WT mice. There was no statistically significant difference. Microscopic examination of the brain at the end of the study revealed that AAV 9.CB7.hCLN2-treated TPP1 m1J In the cortex and hippocampus of knockout mice A decrease in astrocytosis was demonstrated.

[0367] The study aims to improve disease survival and reduce disease-related neurobehavioral deficits, namely circadian activity. normalization of motility and nocturnal activity (which correlates with a decrease in neuroinflammation (astrocytosis)) Based on the results, the minimum effective dose was 3 × 10 11 GC / animal (7.5×10 11 GC / g The brain weight was assumed to be 0.4 g.

[0368] d) AAV9 delivery in C57BL / 6 TPP1m1J knockout mice (I Effect of TPP1 substitution using CV) The goal of this study is to extend lifespan, reduce lysosomal storage material, and eliminate the TPP1 enzyme. Establishing the minimum effective dose of AAV9.CB7.hCLN2 to reduce brain pathology in mice with HIV-1 infection The goal was to achieve this. m1J Groups of knockout mice were treated with vehicle or AAV9 A single ICV injection of .CB7.hCLN2 was administered and survival was monitored. An additional group of 7B1 / 6 mice was included as a control. The study was divided into two parts: The first part evaluated pharmacodynamics (TPP1 activity) and neuropathology after 9 weeks, and the second part evaluated pharmacodynamics (TPP1 activity) and neuropathology after 9 weeks. Part 2 was to evaluate the lifespan of these mice. m1J knock out Groups of mice (n=30 / sex / group) were treated with 0 (vehicle), 1.25 × 10 10 , 5×10 10 , 2 × 10 11 , 8.5×10 11 AAV9.CB7.hCLN at a dose of GC / animal Animals (5 animals / sex / group) were administered a single ICV injection (5 μL) of 2. 9 weeks after administration, The mice were euthanized at 13 weeks of age. The effect of AAV9.CB7.hCLN2 on lifespan was evaluated. Animals assigned to the second part of the study to administer the steroids are ongoing and are described below. The following parameters and endpoints were evaluated: mortality, clinical findings, body weight, and neurological outcomes. Behavioral observations (before administration and at 8 weeks), TPP1 activity, anti-TPP1 antibodies, macroscopic autopsy findings, Organ weights and neuropathology (week 9). Neuropathological examination revealed no significant changes in the brain, cervical spine, and lumbar spinal cord. Loss of ATP, as well as intralysosomal accumulation of autofluorescent storage materials, astrocytosis (GFA P) and microglial activation (CD68) were assessed.

[0369] Vehicle and AAV9.CB7.hCLN2 were administered by ICV injection once on day 1. Untreated TPP1 m1J Clinical signs observed in knockout mice began to appear after 8 weeks. Commonly observed symptoms include tremors and splayed hind legs, which are commonly associated with this disease model. Mortality in this group was 11–18%, similar to previous studies using these mice. As shown in Figures 12A to 12B, the age of ≤5 × 10 10 G The dose of 1.25 × 10 C / animal did not improve survival. 10 In GC / animals, The median survival time was 15 and 17 weeks in males and females, respectively. 10 GC / In animals, median survival times were 18 and 16 weeks for males and females, respectively. 10 11 At the GC / animal dose, a clear improvement in survival was observed in animals now 36 weeks old. Ta.

[0370] At 40 weeks of age, 2 × 10 11 5 / 5 males and 3 / 5 females survived in GC / animal, resulting in an 8.5× 10 11 100% of males and females survived in GC / animals. The clinical signs observed in treated animals were significantly higher than those observed in untreated TPP1 m1J Equivalent to knockout mice Additional findings include ataxia, teeth clicking, and high and low carrier status. At 12 weeks of age (week 8), neurobehavioral observations revealed that AAV9.CB7.hCLN2 In treated animals, untreated TPP1 m1J Field observations not recorded in knockout mice Mild clonic movements and abnormal gait were noted at 20 weeks of age. The majority of animals in the two AAV9.CB7.hCLN2-treated groups showed clonic behavior. Two animals showed convulsions when removed from their home cages. The effect was also observed in 2 / 5 untreated female WT animals at 16 weeks of age. ≧2×10 11 The body weight of GC / animal was similar to that of WT animals in males and slightly lower in females. In the week 9 cohort, untreated or AAV9.CB7.hCLN2 There were no effects on gross findings or organ weights in any of the treated animals.

[0371] At 9 weeks, a dose-related increase in the transgene product (TPP1 activity) was observed in the brain (Figure 13A- 13B), spinal cord (Fig. 14), liver (Figs. 15A-15B), and serum (Figs. 16A-16B). In general, TPP1 activity was greater in males than in females, except in the spinal cord. The majority of AAV9.CB7.hCLN2-treated animals were positive for anti-TPP1 antibodies . High dose (8.5×10 11 GC / animal), compared with a lower dose (1.25 × 10 10 The highest ATPA response was observed in the GC / animal group. The difference between the low and high doses was This may be due to the assay being performed at a high transgene product concentration or may reflect the induction of immune tolerance. Its significance is unclear, as it may be

[0372] Neuropathological analysis correlates with ongoing survival ≥ 2 × 10 11 GC / in animal brain Astrocytosis (Fig. 17A-17B) and microglia in the mouse cortex and thalamus. The decrease in activation of the retina (Figures 18A-18B) was observed. D is 2 x 10 11GC / animal (5×10 11 GC / g brain (assuming brain weight 0.4g) was considered to be equivalent to a dose of

[0373] This study demonstrated that AAV9.CB7.hCLN2 transduces and inhibits the brain, spinal cord, and liver. The subsequent neuropathological hallmarks of CLN2 disease are astrocytosis and microglial activation. It has been shown to improve survival in a mouse model of CLN2 disease through reduced activation. 5×10 10 Animals given the GC / animal dose had similar levels of TPP as those at the highest dose. 1 activity, but there was no improvement in survival rate in groups with equivalent levels of brain TPP1 activity. , survival rate (5×10 10 GC / animal) and its effect on the improvement of neuropathological endpoints This suggests that spinal cord and liver transduction contributes to the overall survival of these animals. This may suggest that

[0374] This study demonstrated improved survival and reduced astrocytosis and microglial activation. Based on this, the minimum effective dose is 2 x 10 11 GC / animal (5×10 11 GC / g brain (brain weight 0. It was assumed to be equivalent to 4g.

[0375] 2. Safety Pharmacology Central nervous system: Neurobehavioral endpoints were measured in a 3-month toxicity study in mice and in cynomolgus monkeys. The study was included in a 4-week pharmacodynamic study in monkeys.

[0376] Three-month toxicity study in mice: Functional Observation Board (FOB) evaluation was performed at 13 weeks. This includes activity, posture, rearing, behavior, and response to stimuli (approach, click, tail). (pinch, and touch), pupillary response, grip response and pain perception (nociceptive [heat]) The study included assessment of response latency to stimuli. These parameters were not affected. , and the number of rearing counts in the field for males is ≥ 2.00 × 10 11 In GC / animals, 8.50 × 10 for females 11 The only notable finding was a decrease in GC / animals In the absence of other findings, this was not considered to be AAV9.CB7.hCLN2-related. It was not considered.

[0377] 4-week pharmacodynamic study in cynomolgus monkeys: In a 4-week study in cynomolgus monkeys, In addition, a comprehensive neurological examination was performed at 4 weeks, which included general sensation and movement. Motor function, cerebral reflexes (pupil, ring and corneal reflexes) and spinal reflexes (sensory reflexes, knee reflexes, cutaneous reflexes) These endpoints included proprioceptive reflexes, tail reflexes, and proprioceptive responses. There were no CB7.hCLN2-related effects, and all animals showed no behavioral or clinical signs during the study. There was no weather.

[0378] Respiratory System: No specific respiratory studies were performed, but after 4 or 13 weeks Respiratory endpoints assessed in the FOB mouse toxicity study, or pulmonary Microscopic changes were unaffected.

[0379] Cardiovascular: No specific cardiovascular studies were performed, and no studies were performed on the effects of treatment with AAV9.CB7.hCLN2. In mouse toxicity studies after 4 or 13 weeks, microscopic changes indicative of cardiovascular effects were not observed. There was no transformation.

[0380] a) Overview TPP1 is a disease model for LINCL-Batten disease (CLN2) m1J Knocka A series of in vivo pharmacological studies were performed using mice. m1J Knock The CLN2 mice have a single nucleotide mutation at the splice donor site downstream of exon 8 of the CLN2 gene. These mice carry a mutation in the gene encoding the soluble lysosomal enzyme TPP1. The natural history study used demonstrated clinical symptoms at an early age, similar to other mouse models of CLN2 disease. The onset of encephalopathy, rapid progression of the abnormal phenotype, and shortened life span, as well as similar pathophysiological and biological It showed the characteristics of CLN2 disease in humans, including chemical and functional changes (Sle at DE,et al. J Neurosci,2004;24(41):9117 -26.;Geraets et al,2017. PLoS One;12(5): e0176526). AAV9.CB7.hCLN2 was transfected with ICV TPP1 m1J knock When administered to mice, the median survival time increased from 18 weeks (untreated) to 40 weeks (tested). Behavioral endpoints were assessed in mice treated with AAV9.CB7.hCLN2. TPP1 m1J Improved motor coordination and learning, as well as clear The recovery of biphasic circadian motor characteristics was observed. 9. There was no significant difference in onset and clinical signs (tremors and abnormal gait) among CB7.hCLN2-treated animals. There were no differences, but there was no progression of these clinical findings and the animals showed signs of physical deterioration as the study continued. In all pharmacological studies, AAV9.CB7.hCLN2 , increased TPP1 activity in the brain and spinal cord. The efficacy of steroids was confirmed by a clear dose-response relationship in terms of survival and reduction in neuropathological endpoints. In all studies using AAV9.CB7.hCLN2, The results showed a clear reduction in lysosomal storage material, normal neuronal morphology, and astrocyte The decrease in CLN2 expression was observed through the expression of TPP1. The efficacy of AAV9.CB7.hCLN2 in attenuating the transpathological phenotype was comparable to that of rhT PP1 to CLN2 - / - After administration to mice and TPP1-null dachshunds, The effects are comparable to those reported in previous studies (Chang et al., 2008; Vuillemeno In addition to increased brain activity, AAVrh10.hCLN2 Hepatic TPP1 activity was increased, suggesting that vasoconstriction may be a contributing factor (Sondhi et al., 2007). The liquid-brain barrier likely prevents TPP1 from leaving the CNS, resulting in systemically detected activity. This may be related to leakage of AAV9.CB7.hCLN2 into the peripheral circulation shortly after administration. Systemic TPP1 may contribute to the accumulation of lysosomal storage materials in tissues and organs outside the CNS. This may be potentially beneficial for the treatment of CLN2 disease, as it may attenuate the dysfunction associated with (Katz,et al,Gene therapy 2017 Feb 24(4): 215-223). Pharmacological studies in rodents on hepatic transduction efficiency (TPP1 activity) The sex-related differences observed in were independent of serotype, promoter, or transgene. , which is thought to be an androgen-dependent effect on AAV uptake (Lonni ng et al,2002.Molecular Therapy Vol.5,No .5;Davidoff et al,2003. Blood.15;102(2): 480-8). A 4-week pharmacodynamic study in cynomolgus monkeys using AAV9.CB7.HCLN2 In this study, there was no clear difference between males and females in liver transduction.

[0381] In summary, AAV9.CB7.hCLN in biologically relevant animal models of disease A single dose of 2 improved survival through increased TPP1 activity in the brain, spinal cord, and liver. Enhanced neuronal loss and autofluorescence storage in the brain, cervical spinal cord, and lumbar spinal cord attenuating intralysosomal accumulation of phospholipids, astrocytosis, and microglial activation In these studies, the minimal effective dose was ≥ 2 × 10 11 GC / animal dose This is thought to be 5 × 10 11 Equivalent to GC / g brain.

[0382] B. Pharmacokinetics and Product Metabolism in Animals Pharmacodynamics (transgene product) and immunogenicity were evaluated using a 3-month toxicity study in mice and a 3-month study in crabs. It was evaluated in a 4-week pharmacodynamic study in monkeys.

[0383] 1. AAV9.CB7.hCLN2: 3-month toxicity study in C57Bl / 6 mice Groups of C57Bl / 6 mice (n=30 / sex / group) were administered 0 (vehicle), 1.25x1 0 10 , 5×10 10 , 2 × 10 11 , 8.5×10 11 AAV9 at a dose of GC / animal. A single ICV injection (5 μL) of CB7.hCLN2 was administered. Recombination studies showed some vector loss at low doses, therefore, 1.25x 10 10 , 5×1010 , 2 × 10 11 , 8.5×10 11 GC / animals to be administered The actual doses were 0.9 x 10 9 (70% recovery), 3.9 × 10 10 (77% Recovery), 1.8 x 10 11 GC / animal (90% recovery) and 8.5 × 10 11 GC / Animal( (100% recovery).

[0384] The results are shown in Figures 19A-19B, 20A-20B, and 21A-21B. Administration of AV9.CB7.hCLN2 was not associated with sex-related differences in brain TPP1 activity. without any side effects, produced a dose-dependent increase in brain TPP1 activity in both males and females (Figures 20A-20C). B). There was no difference in brain TPP1 activity between weeks 4 and 13 in both sexes. Liver T PP1 activity increased in both males and females at both 4 and 13 weeks, and only in males at 4 and 13 weeks. However, in females, hepatic TPP1 activity was significantly elevated at 4 weeks. The mean ... 11 GC / Animal In the 13-week study, liver TPP1 activity was 18 times (males) and 4 times (females) higher than brain TPP1 activity. ) was high. A dose-dependent increase in serum TPP1 activity was observed in both males and females (Fig. 19A-1). 9B). At week 13, serum TPP1 activity was more prevalent in males than in females in all dose groups. High, 8.5 x 10 11 In GC / animal, there was an approximately 11-fold increase between males and females. Serum TPP1 activity at 13 weeks was increased in males and decreased in females compared with that at 4 weeks. .

[0385] The majority of AAV9.CB7.hCLN2-treated animals showed a significant improvement in seroconversion at weeks 4 and 13. The patient tested positive for anti-TPP1 antibodies. 11 GC / animal) , low dose (1.25×10 10 A higher ATPA response was observed in the GC / animal Ta.

[0386] 2. AAV9.CB7.HCLN2: Single-dose intrathecal administration in cynomolgus monkeys Pharmacodynamics study of Groups of cynomolgus monkeys (1 male and 2 females / group) were administered 0, 3.4 × 10 11 , 3.2 × 10 12 or 2.9 x 10 13 The dose is 1 mL / animal (genome copies / GC). AAV9.CB7.hCLN2 was administered by intrathecal injection via cisternal puncture (CM). An additional group of animals (one male and two females) underwent lumbar intrathecal puncture (IT-L; 1 mL / animal). 3.2 x 10 through 12 GC / animal. Serum and CSF samples were collected during the study. TPP1 activity and TPP1 concentration were assessed in serum and CSF (pre-treatment [ -1 or 1 day], 4, 8, 11, 15, 18, 22, 25 and 29 days), liver , spinal cord (cervical, thoracic and lumbar) and brain. For the brain, the frontal cortex, occipital cortex, cerebellum, striatum Superficial or deep samples from the corpus, medulla oblongata, midbrain and thalamus were analyzed.

[0387] Six of the 15 animals had anti-transgene product (TPP1) antibodies (AT On day 29, the incidence of ATPA-positive animals was 0.05%. C / animal = 1 / 3 animal, 3.4 × 10 11 2 / 3 animals in GC / animal (CM), 3.2×1 012 3 / 3 animals in GC / animal (CM), 2.9 × 10 13 GC / animal (CM) 3 / 3 Animals and 3.2 x 10 12 GC / animal (IT-L) was 2 / 3 animals.

[0388] 2.9×10 13 In GC / animals (CM), TPP1 activity and concentration were significantly increased in both serum and CSF. Increased levels were observed, with peak levels generally observed at day 15 (Figures 22A-22B and 23A-23B). TPP1 activity and concentration then declined toward the end of the study. The decrease in serum and CSF TPP1 activity and concentration was consistent with these findings at the end of the study. The immune response observed in animals and commonly observed in non-human primates administered recombinant human proteins was associated with virulence. 12 GC / animal (CM), again likely human derived The decrease in serum serotonin levels after day 18 was associated with the immunogenicity of the transgene product compared to baseline values. There was a minimal increase in mean serum TPP1 activity and concentration in CSF observed in this group. There was no apparent increase in TPP1 activity or concentration, except for one animal, which showed a significant increase between days 8 and 25. There was a slight increase between 3.4×10 11 GC / animal (CM) serum or CSF No increase in TPP1 activity or concentration was observed in the control group.

[0389] 2.9×10 13 GC / animal (CM), midbrain (deep layer [1 / 3 animal] and superficial layer), medulla oblongata Mean TPP compared to controls for the cerebellum (deep and superficial) and cerebellum (deep and superficial) At this dose, the activity of α-glucan-1 was increased in the frontal cortex (deep and superficial layers), striatum (deep and superficial layers), and ), thalamus (superficial), midbrain (deep and superficial), occipital cortex (deep and superficial), medulla oblongata (deep and superficial layers) and cerebellum (deep and superficial layers; Table 3; Figures 24A-24D and 25A-25C) The mean TPP1 concentration was increased compared to the control group. 12 GC / Animal There was no clear difference in TPP1 activity or TPP1 concentration when compared to the control group. Animals showed small trends of elevation in most deep brain regions when compared to the control mean. The lowest dose (3.2 × 10 12 GC / animal) compared with the control group When steroids were administered, increases were observed in all brain regions, but there was no dose-response relationship and generally The relationship to treatment is unclear because values ​​were low compared to dose. Table 3. TPPs in brain regions of AAV9.CB7.hCLN2-treated (CM) cynomolgus monkeys 1 concentration [Table 3]

[0390] 2.9 × 10 compared with the control group 13 In GC / animals, TPP1 activity was increased in all parts of the spinal cord. In 2 / 3 of the animals, these changes were The remaining animals in this group still had elevated TPP1 activity, up to 5.3 times greater than in the control group. Although they showed an increase, this was less than the other two animals and was up to 2.4 times greater than the control group. The increase in TPP1 concentration was in line with the characteristics of TPP1 activity and was observed in all regions of the spinal cord (cervical, Increases in TPP1 activity were observed in the spinal cord (thoracic, lumbar). However, TPP1 concentrations were higher in the lumbar region of the spinal cord compared with the cervical or thoracic regions. .2×10 12 In GC / animals, TPP1 activity was increased in all regions of the spinal cord (cervical, thoracic, and lumbar). The increase was up to two-fold compared to the control group (Figs. 26A-26B). This was also reflected in the 1 concentration, with a maximum 1.8-fold increase observed. 11 GC / animal TPP1 activity and TPP1 concentration were significantly elevated in the cervical, thoracic, and lumbar regions of the spinal cord compared with the control group. The increase was in the region of 1.6 times.

[0391] 2.9×10 13 In GC / animal, TPP1 activity and TPP1 concentration increased 7-fold and 10-fold, respectively. A four-fold increase: 3.2 × 10 12 In GC / animal, TPP1 activity and TPP1 concentration were The increase was 3.7 times and 1.5 times, respectively. 11 In GC / animal, TPP1 activity and TPP1 concentrations increased 1.8-fold and 1.6-fold, respectively.

[0392] Over the course of the study, compared with the control group, 12 GC / Animal(IT -L), there was no clear difference in serum TPP1 activity. This was not reflected in the results because only a slight increase was observed from day 4 onwards until the end of the study. In the case of CSF, TPP1 activity was comparable to that in the control group, but significantly increased compared with baseline. In the CM group, TPP1 levels showed a slight increase from day 8 onwards, which was similar to that observed in the CM group administered at the same dose. No changes were observed in the frontal cortex (deep and superficial layers), striatum (deep and superficial layers), thalamus (deep layers), and superficial layers), midbrain (deep and superficial layers), occipital cortex (deep and superficial layers), medulla oblongata (deep and superficial layers) and the cerebellum (deep and superficial layers), a trend toward minimal increases in TPP1 activity was observed. This was also observed, albeit to a lesser extent, for TPP1 levels, which were mainly observed in single or multiple mice. The increase was due to a slight increase in two animals. Since this was not observed in animals, the relationship to treatment is unclear. There was no clear difference between animals receiving AAV9.CB7.hCLN2 via CM or IT-L at the dose In general, both TPP1 activity and concentration were significantly higher than those in the CM group at the same dose. In the case of IT-L-treated animals, the spinal cord was significantly larger, especially in the cervical and lumbar regions. The increase was related to the fact that animals in this group were placed in the Trendelenburg position immediately after dosing. There is a possibility that this may occur.

[0393] 3. Overview Evaluation of TPP1 (transgene product) in a 3-month mouse toxicity study demonstrated that TPP1 significantly increased the risk of brain, liver, and kidney damage. A dose-related increase in TPP1 activity in serum and in serum was observed. Hepatic TPP1 activity was greater in males than in females, but this was not clear in cynomolgus monkeys. There was no clear difference between 1 month and 3 months, and transgene expression peaked by the 4th week. This study demonstrated that anti-TPP1 activity was significantly improved, as observed in pharmacological studies. The incidence of antibodies was high, with the lowest levels observed at the higher doses, which was attributed to assay interference. This may be due to a decrease in the number of immune cells involved, or may reflect the induction of immune tolerance.

[0394] In a 4-week pharmacodynamic study, AAV9.CB7.hCLN2 administration at a dose of 2.9 × 1 0 13 GC / animal (4.20×10 11 Group GC / g brain (based on brain weight at end of study) induced increased TPP1 activity and concentration in serum and CSF at doses equivalent to the average The decrease in TPP1 observed in CSF and serum by week 2 was consistent with preclinical studies using biotherapy. Similar to the effects generally observed in studies, there are concerns about immunogenicity to the human transgene product. Decreased TPP1 levels were observed in CSF and serum, whereas anti-Tpp The presence of antibodies may affect TPP1 levels in the brain, potentially resulting in increased clearance or other mechanisms. It is unclear whether concentrations may have been underestimated due to mechanisms (neutralizing antibodies) is.

[0395] At the highest dose in this study, recombinant human TPP1 (rhTPP1 ) as well as those published for (Vuillemenot et al., 2014) and increased TPP1 levels in brain regions at day 29. AAV9.CB7 when administered ICV The main difference between rhCLN2 and rhTPP1 is that AAV9.CB7.hCLN2 transduced The cells were administered rhTPP1 by continuous infusion to achieve zero-order pharmacokinetic properties. Therefore, AAV9.CB7.hCL Administration of N2 resulted in increased activity in the brain and CS, as observed with ICV delivery of rhTPP1. There is no such peak-trough pattern for TPP1 in F. The lack of clear effects at low doses is due to the lack of evidence that TPP1 is increased in the brain. The minimum dose in macaques was 4.20 × 10 11 AAV9.CB7.hC in GC / g brain This study suggested that spinal cord transduction may be involved in LN2. The two animals with the greatest TPP1 activity / concentration in the sham rats also had the highest CSF TPP1 activity. This is likely due to the fact that these animals were also observed to have a TPP concentration of 1 in the CSF. This may explain the increase in TPP1. m1JPharmacological studies of knockout mice Similarly, the transgene product increased in the liver and serum. This is related to the distribution of AAV9.CB7.hCLN2 to the annulus, and as previously mentioned, CLN2 disease This study did not show any clear effect at low doses, so Data from rhesus monkeys showed that TPP1 activity increased in the brain and spinal cord by 2.9× 10 13 GC / animal dose (4.20 × 10 11 GC / g brain dose equivalent (at the end of the study) Doses calculated from brain weight may be required, and these doses are This suggests that the dose was equivalent to the minimum effective dose achieved in physical trials.

[0396] C. Toxicity 1. Single-dose study a) AAV9.CB7.hCLN2: 3-month toxicity study in C57Bl / 6 mice The objective of this study was to evaluate the efficacy and safety of AAV9.CB in C57Bl / 6 mice after a single ICV administration. 7. The objective of this study was to evaluate the pharmacodynamics, toxicity, and immunogenicity of hCLN2. Groups of mice (n = 30 / sex / group), 0 (vehicle), 1.25 × 10 10 , 5×10 10 , 2 × 10 11 , 8.5×10 11 A single ICV dose of AAV9.CB7.hCLN2 at a dose of GC / animal injection (5 μL). After 4 weeks (10 animals / sex / group) or 13 weeks (10 Animals were euthanized in groups of 10 (n = 5 / sex / group). An additional group of satellite animals (n = 5 / sex / group) was euthanized. ) were euthanized at each time point to evaluate the transgene product (TPP1 activity) in the brain and liver. Compatibility studies using precision administration devices have shown some vector loss at lower doses. The loss is shown, therefore, 1.25 x 10 10 , 5×10 10 , 2 × 10 11 , 8.5× 10 11 The actual dose administered for GC / animal was 0.9 × 10 9 (70 % recovery), 3.9 x 10 10 (77% recovery), 1.8 × 10 11 GC / animal (90% recovery ) and 8.5 × 10 11 GC / animal (100% recovery).

[0397] The study evaluated the following parameters and endpoints: mortality, clinical findings, Body weight, food consumption, fundus examination, functional observation board (FOB) assessment, clinical pathology parameters ( Hematology and clinical chemistry), TPP1 enzyme activity (serum, brain, and liver), and anti-TPP1 antibodies (blood (clear), gross necropsy findings, organ weights, gross and microscopic examinations.

[0398] Up to 8.5 x 10 over 90 days 11 A single ICV dose of AAV at GC / animal dose There were no clear treatment-related findings in mice given 9.CB7.hCLN2. 10 11 3 of 60 animals administered AAV9.CB7.hCLN2 with GC / animal Death was confirmed within one week after administration (2 / 3) or euthanized immediately before death. (1 / 3), but the cause of death was not determined. Bed signs include thin appearance, hypoactivity, ataxia, cold to the touch, hunched posture, piloerection and heavy breathing. There was some dehydration, as well as stress, suggesting that the animals did not fully recover from the ICV injection. All other microscopic changes in this animal were observed at 4 weeks. These deaths occurred only at the higher doses. Therefore, the relationship with processing is unclear.

[0399] Following FOB assessment at 13 weeks, a significant decrease in field rearing was observed at 13 weeks. Week ≥ 2.00 × 10 in males 11 GC / animal, and 8.50 × 10 in females. 11 GC / Dynamic At 4 weeks, hematological changes (MCV, hematocrit) and M Increased CHC and RDW) and clinical chemistry (urea nitrogen, creatinine, and triglyceride concentrations) There was a small effect on the parameters of sedation (reduction in sedation), which partially resolved by week 13. 8.50x10 11 In GC / animals, there was a decrease in albumin (× ) in males and females at 4 weeks. 0.9) was observed, and at 13 weeks was observed only in females.

[0400] Microscopic changes were observed in the brain, spinal cord, and sciatic nerve after administration of AAV9.CB7.hCLN2. It was 8.50×10 11 GC / animal only, unilateral brain necrosis (at the presumed injection site) and Associated inflammation (subacute / chronic) occurred in 1 / 10 males and 3 / 10 females at week 4 (mild at 13 weeks in 1 / 10 males and 2 / 10 females (minimal to moderate), 2.00×10 11 (2 / 10 males and 2 / 10 females) and 8.50 x 10 11 ( 3 / 10 males and 7 / 10 females) GC / animal, peripheral blood mononuclear cell infiltration into the mouse brain at 4 weeks. At week 13, the mean value was 8.50 × 10 11 GC / animals only These infiltrates were observed in a small number of mice (1 / 10 males and 5 / 10 females) at 100°C. , inflammation, and / or an immune response to the test article.

[0401] In the spinal cord, peripheral nerve root axonal degeneration (minimal to moderate) is ≥ 2.00 × 10 11 In GC / animals, axonal degeneration was observed in mice at 4 and 13 weeks. Symptoms (minimal to moderate) are ≥ 5.00 × 10 10 GC / animals at weeks 4 and 13 Minimal axonal degeneration of the sciatic nerve was also observed in males sacrificed on day 4. These findings were present in multiple nerve sections, which suggests that the findings were bilateral. Axonal degeneration is characterized by eosinophilic axonal swelling, axonal fragmentation, and phagocytic myelin. characterized by the formation of digestive chambers with phage and associated reactive gliosis .

[0402] Non-adverse findings associated with AAV9.CB7.hCLN2 were observed at weeks 4 and 13. ≧1.25×10 10 In GC / animals, minimal to mild lymphoid hyperplasia occurred in the spleen of mice. formation (germinal centers and marginal zone), and hyperplasia of the red pulp, hematopoietic cells, and / or marginal zone ( The findings consisted of an increased incidence and / or severity of cellularity in the thyroid gland (minimal to mild). These findings correlated with an increase in spleen weight. At 13 weeks, the changes in the spleen were It was not detected in male animals only at the high dose.

[0403] Based on findings in the sciatic nerve and spinal roots, a no observed adverse effect level (NOAE) was determined. L) is 1.25 x 10 10 It was considered to be GC / animal.

[0404] b) AAV9.CB7.hCLN2: Single-dose intrathecal administration in cynomolgus monkeys Pharmacodynamics study of The objective of this study was to evaluate the efficacy and safety of AAV9.CB7.hCLN2( The objective of this study was to evaluate the pharmacodynamics and toxicity of AAV9.hCLN2. 1 male and 2 females / group) were administered 0, 3.4 × 10 11 , 3.2 × 10 12 or 2.9 x 10 13 via cisternal puncture (CM) at a dose of 100 genome copies (GC) / animal (1 mL / animal). AAV9.CB7.hCLN2 was administered by intrathecal injection. Two males and two females received 3.2 × 10 12 GC / animal. At the end of the study, animals were euthanized on day 29. Key points: clinical findings, weight, food consumption (qualitative), neurological examination (general sensation and movement) motor function, cerebral and spinal reflexes), pharmacodynamics (TPP1 activity and concentration), CSF total cell count, organ Weight, macroscopic and microscopic examination (brain [spanning the forebrain to the brainstem], spinal nerve roots and ganglia) Attached spinal cord (cervical, thoracic, lumbar and lumbosacral regions), sciatic nerve and trigeminal ganglion). For titration, serum, CSF, liver, spinal cord (cervical, thoracic and lumbar regions) and brain (frontal cortex, We analyzed superficial and deep samples from the occipital cortex, cerebellum, striatum, medulla oblongata, midbrain, and thalamus. The samples were evaluated.

[0405] Treatment-related clinical signs, effects on body weight, or physical and neurological symptoms observed during the study There were no significant changes in CSF cell counts at the end of the study.

[0406] 2.9×10 13In GC / animal (CM), dorsal root ganglia (DRG), mainly 2 / 3 of animals There were microscopic changes observed in the lumbosacral DRG. The main changes were neurodegeneration (minimal to mild) and increased cellularity, mainly in the spinal nerve roots and dorsal Nerve roots (1 / 3 animals only) and dorsal spinal cord in the lumbar (moderate) and lumbosacral (moderate) regions Lymphocyte / macrophage infiltration (moderate to marked) associated with increased severity of tract degeneration In a single animal at this dose, microscopic changes in the brain (neurodegeneration, gliosis) were also observed. Although it was thought to be potentially harmful, the changes in the brain were minimal and did not cause any impairment of the nervous system. No harm was expected. There was no evidence of glial reactivity, and other animals in this study Neurodegeneration in the brain was therefore unlikely. Necrotic neurons were quickly removed. However, there was usually a detectable persistent glial response in the two affected animals. , findings in the DRG indicated a higher degree of transduction (TPP) compared to unaffected animals. The TPP1 activity in the lumbar region of these animals was associated with was more than two times greater than in unaffected animals.

[0407] 3.2×10 12 In GC / animal (CM), the lumbosacral DRG was predominant in one-third of the animals observed. Microscopic changes were observed in the DRG. The changes in the DRG were characterized by neuronal degeneration (moderate). and increased cellularity, primarily in the lumbar and lumbosacral spinal nerve roots (moderate) and in the lumbosacral spinal nerve roots (moderate). Lymphocyte / macrophage infiltration is associated with increasing severity of dorsal nerve root (moderate) degeneration (Notable).

[0408] 3.2×10 12In GC / animal (IT-L), changes in the DRG were observed in the lumbosacral region (mild ) and increased cellularity in the lumbar spine and lumbosacral region (moderate). In one animal, the cervical part of the DRG was infiltrated with lymphocytes / macrophages. Significant increases in solidity and minimal neuronal degeneration were observed. Increased severity of lumbosacral spinal nerve root degeneration (moderate) and cervical dorsal nerve root degeneration (mild) The changes in the neck of these animals were more pronounced in the neck compared to the IT-CM group. This may be related to a high degree of transduction (i.e., TPP1 activity and concentration). This animal showed the highest TPP1 activity in the neck, approximately 10% higher than the other two animals in this group. In this group, the two animals with the highest TPP1 activity in the lumbar region of the spinal cord It is also noted that animals were observed with degeneration in the lumbar / lumbosacral DRG.

[0409] 2.9×10 13 or 3.2 x 10 12 observed in animals treated with GC / animal Degeneration of the spinal nerve roots, dorsal nerve roots, and dorsal spinal tracts associated with IT catheter placement While this may be due to the presence of ATP, it is not observed in control animals and is not a partial explanation for the degeneration at this level. This is due to increased cellularity and neuronal degeneration in the DRG of some ganglia. there was.

[0410] The exact cellular components of the increased cellularity observed in some DRGs remain to be determined. Morphological appearance showed an increase in satellite glial cells and lymphocytes and macrophages. The combination of phage infiltration and IBA-1 staining of spinal cord sections (for microglia) was suggested. ) revealed infiltrating macrophages in several ganglia present in spinal cord sections from multiple animals. The major cell type comprising the increased cellularity was infiltrating lymphocytes. There was no clear correlation between ATPA-positive animals and the occurrence of findings in the DRG. At the highest dose, all animals were ATPA positive, but DRG changes were Only 2 / 3 animals were observed. However, 3.2 × 10 12 GC / Animal(IT- The one animal that tested negative after administration of L) showed no DRG findings.

[0411] 3.4×10 11 No test article-related changes were observed in GC / animals. Therefore, based on findings in DRG, the no observed adverse effect level (NOAEL) was 3.4 × 10 11 It was thought to be GC / animal.

[0412] 2. Genotoxicity AAV9.CB7.hCLN2 is a recombinant AAV that does not contain the integration machinery of WT AAV. V. Once AAV is internalized into the host cell, it transports to the nucleus and uncoats. AAV9.CB7.hCLN2 undergoes a process called cloning to form a circular episome that persists in the nucleus. The source helper plasmid used for production contains essential cis-elements important for replication. Since the gene does not contain the cleavage site, it cannot replicate in the transduced cells. In addition to the presence of the rep element, it also requires an active helper virus to reproduce. This is in contrast to WT AAV, which is

[0413] Based on the available scientific literature and clinical data, AAV integration has not been observed. Although there are several articles suggesting an increased incidence of liver tumors in AAV-treated mice; Vector DNA was largely absent in these tumors and lacked expression of the transgene product ( Bell P, et al. Mol Ther 2006;25:34-44). recently study, tumor incidence was significantly higher in AAV9-treated MPS II mice than in untreated WT mice. The results were very similar in mice (38% and 36%, respectively) (Fu H, et al. ol Ther Methods Clin Dev.2018;10:327-340 Therefore, the recently approved AAV9, onasemnogene abeparv Along with ovec-xioi, the weight of evidence from recent studies supports the use of rAAV This confirms the low risk of insertional mutagenicity.

[0414] 3. Carcinogenicity Tests A 3-month mouse toxicity study with AAV9.CB7.hCLN2 showed no pre-neoplastic or neoplastic No neoplastic lesions were observed.

[0415] 4. Other Toxicity Studies a) Immunotoxicity From toxicity study data using AAV9.CB7.hCLN2 in mice and monkeys , showing AAV9.CB7.hCLN2-related effects on leukocytes or immunotoxicity No morphological changes were noted in the spleen, lymph nodes, or thymus (ICH S8 The spleen changes observed in the mouse toxicity study were similar to those observed for the human transgene product. This may be considered an adaptive response to the increased immunogenicity of the antibody.

[0416] b) Overview AAV9.CB7, including pharmacodynamics (transgene product) and immunogenicity (anti-TPP1 antibodies). Non-clinical safety of hCLN2 was evaluated in single-dose toxicity studies in cynomolgus monkeys and mice The studies evaluated AAV9 for up to one month and up to three months, respectively. There were no life-threatening findings associated with CB7.hCLN2. The main findings were axonal degeneration in the spinal cord, dorsal gan...

Claims

1. A pharmaceutical composition formulated for storage in a vial as a frozen solution and for single intrathecal administration, comprising: (a) a recombinant adeno-associated virus (rAAV) comprising an AAV9 capsid and vector genome; (b) sodium chloride; (c) magnesium chloride, (d) potassium chloride, (e) dextrose, (f) poloxamer 188, (g) sodium phosphate at a concentration of about 1 mM, and (h) calcium chloride Including, The pharmaceutical composition does not contain a bicarbonate buffer; The pharmaceutical composition, wherein the pH of the pharmaceutical composition is in the range of about 6.0 to about 7.

5.

2. The pharmaceutical composition of claim 1, formulated for single administration in a vial for storage as a 1 mL frozen solution.

3. A pharmaceutical composition described in claim 1 or 2, wherein the approximately 1 mM sodium phosphate is composed of sodium phosphate monobasic and sodium phosphate dibasic.

4. 4. The pharmaceutical composition of claim 3, wherein the sodium chloride, magnesium chloride, potassium chloride, dextrose, poloxamer 188, monobasic sodium phosphate, dibasic sodium phosphate, and calcium chloride are each independently in the form of anhydrous, monohydrate, dihydrate, trihydrate, tetrahydrate, pentahydrate, hexahydrate, heptahydrate, octahydrate, nonahydrate, or decahydrate.

5. (a) the rAAV; (b) sodium chloride at a concentration of about 150 mM; (c) magnesium chloride at a concentration of about 1.2 mM; (d) potassium chloride at a concentration of about 3.0 mM; (e) calcium chloride at a concentration of about 1.4 mM; (f) dextrose at a concentration of about 4.4 mM; (g) poloxamer 188 at a concentration of about 0.001% (volume / volume); (h) monobasic sodium phosphate at a concentration of about 0.20 mM, and 5. The pharmaceutical composition of any one of claims 1 to 4, comprising (i) dibasic sodium phosphate at a concentration of about 0.80 mM.

6. The vector genome concentration (VGC) of the pharmaceutical composition is about 1 x 10 １１ GC / mL, approximately 3 x 10 １１ GC / mL, approximately 6 x 10 １１ GC / mL, approximately 1×10 １２ GC / mL, approximately 3 x 10 １２ GC / mL, approximately 6 x 10 １２ GC / mL, approximately 1×10 １３ GC / mL, approximately 2 x 10 １３ GC / mL, approximately 3 x 10 １３ GC / mL, approximately 4 x 10 １３ GC / mL, approximately 5 x 10 １３ GC / mL, approximately 6 x 10 １３ GC / mL, approximately 7 x 10 １３ GC / mL, approximately 8 x 10 １３ GC / mL, approximately 9×10 １３ GC / mL, or approximately 1 x 10 １４ GC / mL, approximately 3 x 10 １４ GC / mL, approximately 6 x 10 １４ GC / mL, or approximately 1 x 10 １５ The pharmaceutical composition according to any one of claims 1 to 5, wherein the concentration is GC / mL.

7. 7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the pH of the pharmaceutical composition is in the range of about 6.5 to about 7.

5.

8. The pharmaceutical composition of any one of claims 1 to 7, wherein the pH of the pharmaceutical composition is about 7.2, about 7.3, or about 7.

4.

9. The pharmaceutical composition of claim 1, wherein the vector genome comprises: (i) an AAV 5' inverted terminal repeat (ITR) sequence; (ii) a promoter; (iii) a coding sequence; and (iv) an AAV 3' ITR.

10. The promoter is (a) the chicken β-actin (CBA) promoter, or (b) a hybrid promoter comprising a CBA promoter sequence and a cytomegalovirus enhancer element; The pharmaceutical composition of claim 9, wherein

11. The pharmaceutical composition of claim 9 or 10, wherein the rAAV comprises an AAV 5' ITR and / or an AAV 3' ITR derived from AAV2.

12. The vector genome (a) polyA, and / or (b) an intron, and / or (c) enhancer The pharmaceutical composition according to any one of claims 1 to 11, further comprising:

13. (a) 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); and / or (b) the intron is derived from CBA, human beta globin, IVS2, SV40, bGH, alpha globulin, beta globulin, collagen, ovalbumin, or p53; and / or (c) the enhancer is a CMV enhancer, an RSV enhancer, an APB enhancer, an ABPS enhancer, an αmic / bik enhancer, a TTR enhancer, en34, or an ApoE enhancer; The pharmaceutical composition of claim 12.

14. 14. The pharmaceutical composition of any one of claims 1 to 13, wherein the size of the rAAV vector genome is about 3 kilobases to about 5.5 kilobases, or about 4 kilobases.

15. A pharmaceutical composition described in any one of claims 1 to 14, wherein the rAAV is RGX-121.

16. A pharmaceutical composition described in any one of claims 1 to 15, wherein the rAAV is RGX-111.

17. 17. The pharmaceutical composition of any one of claims 1 to 16, wherein the rAAV is at least 2%, 5%, 7%, 10%, 12%, 15%, 17%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, or 1000-fold more stable to freeze / thaw cycles than the same recombinant rAAV in a reference pharmaceutical composition.

18. The stability of the rAAV is (a) rAAV infectivity; (b) rAAV aggregation levels, or (c) the pharmaceutical composition of claim 17, as determined by the level of free DNA released by the rAAV.

19. The pharmaceutical composition (a) a liquid composition, or (b) a frozen composition, or (c) a lyophilized composition, or (d) Reconstituted Lyophilized Composition The pharmaceutical composition according to any one of claims 1 to 18, wherein

20. 20. The pharmaceutical composition of any one of claims 1 to 19, wherein the pharmaceutical composition has properties that make it suitable for intracerebroventricular (ICV), intracisternal (IC), lumbar intrathecal, intracranial, intravenous, intravascular, intraarterial, intramuscular, intraocular, intramuscular, subcutaneous, or intradermal administration.

21. 21. The pharmaceutical composition of any one of claims 1 to 20, wherein the rAAV is produced using a method comprising growing in suspension culture a suspension cell line capable of producing the rAAV.

22. 22. A kit comprising one or more containers and instructions for use, wherein at least one of the one or more containers contains a pharmaceutical composition according to any one of claims 1 to 21.

23. A method for preparing a pharmaceutical composition according to any one of claims 1 to 21, comprising mixing the rAAV with sodium chloride, magnesium chloride, potassium chloride, calcium chloride, monobasic sodium phosphate, dibasic sodium phosphate, dextrose, poloxamer 188, and water.

24. A method for producing a recombinant adeno-associated virus (rAAV) pharmaceutical, wherein the rAAV comprises AAV capsid serotype 9 (AAV9) and a vector genome, the method comprising: (a) growing in suspension culture a suspension cell line capable of producing the rAAV; (b) recovering the rAAV; (c) clarifying the rAAV harvest by filtration; (d) purifying the rAAV; (e) concentrating the rAAV; (f) formulating the rAAV, comprising combining the rAAV with sodium chloride, magnesium chloride, potassium chloride, calcium chloride, sodium phosphate, dextrose, poloxamer 188, and water to form a pharmaceutical preparation, wherein the pharmaceutical preparation comprises sodium phosphate at a concentration of about 1 mM and is free of bicarbonate buffer; (g) filling the formulated pharmaceutical product into single-dose vials; and (h) storing the vial frozen. The method comprising:

25. The method of claim 24, wherein step (g) comprises filling 1 mL of the formulated pharmaceutical product into a single-dose vial.

26. The method of claim 24 or 25, wherein the formulated pharmaceutical comprises sodium chloride at a concentration of about 150 mM, magnesium chloride at a concentration of about 1.2 mM, potassium chloride at a concentration of about 3.0 mM, calcium chloride at a concentration of about 1.4 mM, dextrose at a concentration of about 4.4 mM, poloxamer 188 at a concentration of about 0.001% (volume / volume), sodium phosphate monobasic at a concentration of about 0.20 mM, and sodium phosphate dibasic at a concentration of about 0.80 mM.

27. A method for producing a recombinant adeno-associated virus (rAAV), wherein the rAAV comprises an AAV9 capsid and a vector genome, the method comprising: (a) growing in suspension culture a suspension cell line capable of producing the rAAV; (b) recovering the rAAV; (c) clarifying the rAAV harvest by filtration; (d) purifying the rAAV; (e) concentrating the rAAV; (f) preparing the pharmaceutical composition according to any one of claims 1 to 21 for frozen storage. The method comprising:

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