AAV vector treatment methods for late infantile neuronal ceroid lipofuscinosis type 2

Abstract

To provide methods for treating a primate in need of tripeptidyl peptidase 1 (TPP1).SOLUTION: The present invention provides methods comprising steps of (a) providing a recombinant adeno-associated virus (AAV) vector comprising a nucleic acid encoding TPP1; and (b) administering a predetermined amount of the recombinant AAV vector to the central nervous system (CNS) of the primate, wherein the TPP1 is expressed in the primate.SELECTED DRAWING: Figure 3B

Description

Background Art

[0001]

[0002] Late infantile neuronal ceroid lipofuscinosis type 2 (CLN2), also known as Jansky-Bielschowsky disease and late infantile NCL (LINCL), is a progressive neurodegenerative disorder that affects children around the age of 2 - 4 years. Symptoms include seizures, loss of motor control and vision, and cognitive and developmental impairments, leading to death within 20 years of birth. The underlying pathological mechanism is a deficiency or defect in the soluble lysosomal enzyme tripeptidyl peptidase-1 (TPP1) due to mutations in the corresponding gene.

[0002]

[0003] Reports have shown that transduction of ependymal cells lining the lateral ventricles of the brain with an adeno-associated virus (AAV) vector provides sustained secretion of human TPP1 into the cerebrospinal fluid (CSF), thereby delivering the TPP1 protein expressed throughout the central nervous system (Martz, L., Biocentury Innovation, December 10, 2015). Delivery of AAV2-CAG-TPP1 via ependymal transduction in a canine model of CLN2 has been reported to provide disease modification and life extension (Katz, M. L. et al., (2015). Sci Transl Med, 7(313)).

Summary of the Invention

[0003]

[0004] This specification discloses non-human primate studies evaluating the safety and tolerability of an AAV2-CAG-human TPP1 vector. The AAV vector was delivered by unilateral injection into the lateral ventricle at three doses in the range of 1E13 - 2.17E14 vector genomes / brain, followed by observations at 5 weeks and 20 weeks. Changes in TPP1 activity and antigen levels in the CSF from baseline in each animal were monitored. TPP1 activity levels showed peak increases of approximately 17-fold in the low-dose cohort and approximately 48-fold in the high-dose cohort compared to baseline. Furthermore, during the study period, the mean transgene expression levels of hTPP1 at all doses tested were at the K of TPP1 取り込み ​It exceeded the range. In preliminary analysis of related central nervous system (CNS) tissues, no pathological changes related to vector delivery or expression of the TPP1 transgene have been identified. In conclusion, expression of human TPP1 after transduction of astrocytes using an AAV2 vector in non-human primates results in sustained CSF TPP1 protein expression within or above the range of about 60 to about 120 ng / mL of K, which is sufficient to confer a therapeutic effect in animals with CLN2. 取り込み It was well tolerated if consistent persistent CSF TPP1 protein expression within or above the 取り込み value was observed.

[0004]

[0005] In one embodiment, a method of treating a primate in need of tripeptidyl peptidase 1 (TPP1) comprising: (a) providing a recombinant adeno-associated virus (AAV) vector comprising a nucleic acid encoding TPP1; and (b) administering a predetermined amount of the recombinant AAV vector to the central nervous system (CNS) of the primate, wherein the TPP1 is expressed in the primate.

[0005]

[0006] In one embodiment, the primate is a human. In one embodiment, the human has late infantile neuronal ceroid lipofuscinosis (CLN2). In one embodiment, the human is about 1 to 10 years old or older than 10 years old. In one embodiment, the human is about 2 to 5 years old.

[0006]

[0007] In one embodiment, in the method of treating a primate, the recombinant AAV vector is administered to the lateral ventricle or the cisterna magna. In one embodiment, the recombinant AAV vector is administered to the occipital horn of the lateral ventricle. In one embodiment, the recombinant AAV vector is administered unilaterally to one lateral ventricle. In one embodiment, the recombinant AAV vector is administered bilaterally to each lateral ventricle. In one embodiment, the recombinant AAV vector is administered unilaterally or bilaterally multiple times to one or both lateral ventricles.

[0007]

[0008] In certain embodiments, TPP1 is expressed at increased levels in the CNS. In certain embodiments, TPP1 is expressed or delivered throughout the CNS. In certain embodiments, TPP1 is expressed or delivered in epithelial cells. In certain embodiments, TPP1 is substantially delivered.

[0008]

[0009] In certain embodiments, TPP1 expression is maintained at levels equal to or greater than those required for maximal uptake of TPP1 into neurons. In certain embodiments, TPP1 expression is maintained at levels 取り込み equal to or greater than K, where K 取り込み is at least about 60 ng / mL. In certain embodiments, TPP1 expression is maintained at levels 取り込み equal to or greater than K, where K 取り込み is at least about 60 ng / mL to 120 ng / mL. In certain embodiments, TPP1 expression is maintained at levels greater than about 120 ng / mL. In certain embodiments, TPP1 expression is maintained at levels greater than about 150 ng / mL, greater than about 200 ng / mL, greater than about 250 ng / mL, or greater than about 300 ng / mL. In certain embodiments, TPP1 expression is maintained in the CNS for at least about 5 weeks, or at least about 10 weeks, or at least about 20 weeks. In certain embodiments, detectable TPP1 expression or TPP1 activity is maintained in the CNS for at least 5 weeks, or at least 10 weeks, or at least 20 weeks.

[0009]

[0010] In certain embodiments, in a method of treating a primate, the recombinant AAV vector is at a dose greater than about 1.5×10 13 AAV vector genomes; at a dose of about 5×10 13 AAV vector genomes or greater than about 5×10 13 AAV vector genomes; at a dose of about 1×10 14 AAV vector genomes or greater than about 1×10 14 AAV vector genomes; at a dose of about 5×10 14 AAV vector genomes or greater than about 5×10 14at a dose of AAV vector genome exceeding; about 1×10 15 AAV vector genome or about 1×10 15 at a dose of AAV vector genome exceeding; or about 5×10 15 AAV vector genome or about 5×10 15 at a dose of AAV vector genome exceeding is administered to the CNS.

[0010]

[0011] In certain embodiments, in a method of treating a primate, the recombinant AAV vector is from about 1.5×10 13 to about 5×10 15 in a dose range of vector genomes; about 1×10 14 to about 3×10 15 in a dose range of vector genomes; about 2×10 14 to about 2×10 15 in a dose range of vector genomes; about 2.5×10 14 to about 7.5×10 14 in a dose range of vector genomes; about 5×10 14 to about 5×10 15 in a dose range of vector genomes; or about 1×10 15 to about 5×10 15 in a dose range of vector genomes is administered to the CNS.

[0011]

[0012] In certain embodiments, in a method of treating a primate, the recombinant AAV vector is at a dose of about 1×10 14 vector genomes, at a dose of about 2×10 14 vector genomes, at a dose of about 3×10 14 vector genomes, at a dose of about 4×10 14 vector genomes, at a dose of about 5×10 14 vector genomes, at a dose of about 6×10 14 vector genomes, at a dose of about 7×10 14 vector genomes, at a dose of about 8×10 14 vector genomes, at a dose of about 9×10 14 vector genomes, at a dose of about 1×10 15 vector genomes, at a dose of about 2×10 15 vector genomes, at a dose of about 3×10 15administered to the CNS at a dose of about 4×10 15 administered to the CNS at a dose of about 5×10 15 vector genomes, or vector genomes.

[0012]

[0013] In certain embodiments, the method reduces, decreases, or suppresses one or more symptoms of CLN2; or prevents or reduces the progression or worsening of one or more symptoms of CLN2; or stabilizes one or more symptoms of CLN2; or improves one or more symptoms of CLN2.

[0013]

[0014] In certain embodiments, the one or more symptoms are selected from the group consisting of visual impairment, cognitive developmental disorder or deficit, loss of motor control, and seizures.

[0014]

[0015] In certain embodiments, the nucleic acid encoding TPP1 comprises an expression cassette operably linked to an expression control element. In certain embodiments, the expression control element is located 5' to the nucleic acid. In certain embodiments, the expression control element comprises a CAG (SEQ ID NO: 3) promoter, a cytomegalovirus (CMV) immediate early promoter / enhancer, a Rous sarcoma virus (RSV) promoter / enhancer, an SV40 promoter, a dihydrofolate reductase (DHFR) promoter, or a chicken β-actin (CBA) promoter.

[0015]

[0016] In certain embodiments, the heterologous nucleic acid is located between one or more 5' and / or 3' AAV inverted terminal repeats (ITRs (plural)). In certain embodiments, the one or more 5' and / or 3' AAV ITRs (plural) include mutated, modified, or variant AAV ITRs that are not processed by the AAV Rep protein. In certain embodiments, the one or more 5' and / or 3' AAV ITRs (plural) include mutated, modified, or variant AAV ITRs that enable or facilitate the formation of a self-complementary reporter transgene genome into a double-stranded inverted repeat sequence structure in a recombinant AAV vector. In certain embodiments, the mutated, modified, or variant AAV ITR has a deleted D sequence and / or a mutated, modified, or variant terminal resolution site (TRS) sequence.

[0016]

[0017] In certain embodiments, the recombinant AAV vector comprises, in the 5'→3' direction, a first AAV ITR; a promoter operable in mammalian cells; a heterologous nucleic acid; a polyadenylation signal; and optionally a second AAV ITR.

[0017]

[0018] In certain embodiments, the one or more ITRs (plural) include AAV serotype AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh74, or Rh10 ITRs.

[0018]

[0019] In certain embodiments, the recombinant AAV vector comprises a VP1, VP2, and / or VP3 that is at least 60% identical to the VP1, VP2, and / or VP3 of AAV serotype AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh74, Rh10, SPK1 (SEQ ID NO: 1), or SPK2 (SEQ ID NO: 2), or a hybrid or chimeric VP1, VP2, and / or VP3 sequence of any of the foregoing AAV serotypes. In certain embodiments, the recombinant AAV vector comprises a VP1, VP2, and / or VP3 capsid protein that has 100% sequence identity to a VP1, VP2, and / or VP3 capsid protein selected from the group consisting of the VP1, VP2, and / or VP3 capsid proteins of AAV1, AAV2, AAV3, AAV3B, AAV, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, Rh74, SPK1 (SEQ ID NO: 1), and SPK2 (SEQ ID NO: 2).

[0019]

[0020] In certain embodiments, the recombinant AAV vector further comprises a polyadenylation sequence located on the 3' side of the nucleic acid. In certain embodiments, the nucleic acid encoding TPP1, the expression control element, or the polyadenylation sequence has a reduced CpG compared to the wild-type nucleic acid, expression control element, or polyadenylation sequence encoding TPP1. In certain embodiments, the polyadenylation sequence comprises a bovine growth hormone (bGH) polyadenylation sequence.

[0020]

[0021] In certain embodiments, TPP1 is human and comprises or consists of the sequence set forth as SEQ ID NO: 4, or a functional variant or polymorphic form thereof.

[0021]

[0022] In certain embodiments, the recombinant AAV vector comprises (a) one or more AAV capsids, and (b) one or more AAV inverted terminal repeats (ITRs (plural)), and one or more AAV ITRs flank the 5' or 3' end of the nucleic acid or expression cassette.

[0022]

[0023] In certain embodiments, the recombinant AAV vector further comprises an intron located 5' or 3' of one or more ITRs (plural available).

[0023]

[0024] In certain embodiments, at least one or more of the one or more ITRs (plural available) and / or introns are modified to have reduced CpG.

[0024]

[0025] In certain embodiments, the recombinant AAV vector comprises a capsid serotype comprising an AAV VP1, VP2, and / or VP3 capsid having at least 90% sequence identity with an AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, AAV-2i8, SPK1 (SEQ ID NO: 1), or SPK2 (SEQ ID NO: 2) VP1, VP2, and / or VP3 sequence; or a capsid having at least 95% sequence identity with an AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, AAV-2i8, SPK1 (SEQ ID NO: 1), SPK2 (SEQ ID NO: 2) VP1, VP2, and / or VP3 sequence; or a capsid having 100% sequence identity with an AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, AAV-2i8, SPK1 (SEQ ID NO: 1), or SPK2 (SEQ ID NO: 2) VP1, VP2, and / or VP3 sequence.

[0025]

[0026] In certain embodiments, one or more ITRs (plural available) comprise one or more ITRs of any of the AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, or Rh74 AAV serotypes, or combinations thereof.

[0026]

[0027] In certain embodiments, the recombinant AAV vector is in a pharmaceutical composition comprising a biologically compatible carrier or excipient.

[0027]

[0028] In certain embodiments, the pharmaceutical composition further comprises empty AAV capsids. In certain embodiments, the ratio of empty AAV capsids to recombinant AAV vectors is in the range of about 100:1 to 50:1, about 50:1 to 25:1, about 25:1 to 10:1, about 10:1 to 1:1, about 1:1 to 1:10, about 1:10 to 1:25, about 1:25 to 1:50, or about 1:50 to 1:100 or therebetween. In certain embodiments, the ratio of empty AAV capsids to recombinant AAV vectors is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0028]

[0029] In certain embodiments, the pharmaceutical composition further comprises a surfactant.

Brief Description of the Drawings

[0029]

Figure 1

Figure 2A

Figure 2B

Figure 3A

Figure 3B

Figure 4

Mode for Carrying Out the Invention

[0030]

[0034] The TPP1 "polypeptide", "protein" and "peptide" encoded by a "nucleic acid" or "polynucleotide" sequence includes the full-length native TPP1 sequence, as well as functional TPP1 sub-sequences, modified forms or sequence variants, similar to the naturally occurring wild-type TPP1 protein, as long as the sub-sequence, modified form or variant retains some functionality of the native full-length TPP1 protein. In the methods and uses of the present invention, such TPP1 polypeptides, proteins and peptides encoded by a nucleic acid sequence can, but need not, be identical to the endogenous TPP1 protein that is defective, or has insufficient expression, or is deficient in the mammal being treated.

[0031]

[0035] The TTP1 polypeptide or TPP1 encoding the polynucleotide may each contain one or more amino acid residues or nucleotide modifications, for example, but not limited to, one or more amino acid residue or nucleotide substitutions (e.g., 1-3, 3-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-40, 40-50, 50-100, 100-150, 150-200, 200-250, 250-500, 500-750, 750-850 or more amino acid residues or nucleotides).

[0032]

[0036] Examples of amino acid modifications are, for example, conservative amino acid substitutions or deletions (e.g., sub-sequences or fragments) of the reference sequence in TPP1. In certain embodiments, the modified or variant TPP1 sequence retains at least a portion of the function or activity of the unmodified TPP1 sequence.

[0033]

[0037] All mammalian and non-mammalian forms of nucleic acid encoding TPP1, including other mammalian forms of TPP1, are expressly included whether known or unknown.

[0034]

[0038] As used herein, the term "vector" refers to a small carrier nucleic acid molecule, plasmid, virus (e.g., AAV vector), or other vehicle that can be manipulated by insertion or incorporation of a nucleic acid. Such vectors can be used for genetic manipulation (i.e., "cloning vectors"), for introduction / transfer of polynucleotides into cells, and for transcription or translation of polynucleotides inserted in cells. An "expression vector" is a specialized vector that contains a gene or nucleic acid sequence having the essential regulatory regions required for expression in a host cell.

[0035]

[0039] Vector nucleic acid sequences generally contain at least an origin of replication for growth in cells, and optionally additional elements such as heterologous nucleic acids (e.g., nucleic acids encoding TPP1), expression control elements (e.g., promoters, enhancers), introns, inverted terminal repeats (ITRs), selection markers (e.g., antibiotic resistance), polyadenylation signals, etc.

[0036]

[0040] Viral vectors are derived from or based on one or more nucleic acid elements containing a viral genome. Specific viral vectors include adeno-associated virus (AAV) and lentiviral vectors.

[0037]

[0041] The term "recombinant" means that a composition has been engineered (i.e., genetically engineered) in a manner that does not generally occur in nature, as a modifier for vectors such as recombinant adeno-associated virus (rAAV) vectors, and for sequences such as recombinant nucleic acids and polypeptides. A specific example of a recombinant AAV vector is when a nucleic acid sequence that is not normally present in the wild-type AAV genome is inserted into the AAV genome. The term "recombinant" is not always used herein with respect to AAV vectors and sequences such as nucleic acids, but recombinant forms containing polynucleotides are explicitly included notwithstanding such an omission.

[0038]

[0042] A "recombinant AAV vector" or "rAAV" is derived from the wild-type AAV genome by using molecular methods to remove the wild-type genome from the AAV genome and replace it with a non-native nucleic acid sequence called a heterologous nucleic acid. Typically, for AAV, one or both inverted terminal repeat (ITR) sequences of the AAV genome are retained in the AAV vector. rAAV is distinguished from the AAV genome because all or part of the AAV genome has been replaced with a non-native sequence with respect to the AAV genomic nucleic acid. Thus, the incorporation of non-native (non-AAV) sequences defines the AAV vector as a "recombinant" vector, which can be referred to as an "rAAV vector".

[0039]

[0043] The rAAV array can be packaged (referred to herein as "particle") ex vivo, in vitro or in vivo for subsequent infection (transduction) of cells. When a recombinant AAV vector array is encapsulated or packaged into AAV particles, the particles can also be referred to as "rAAV vectors" or "rAAV particles". Such rAAV particles contain proteins that encapsulate or package the vector genome, and in the case of AAV, they are referred to as capsid proteins.

[0040]

[0044] What is conveniently abbreviated as "vector genome" or "vg" refers to a portion of a recombinant plasmid array that is ultimately packaged or encapsulated to form viral (e.g., rAAV) particles. When constructing or manufacturing a recombinant vector using a recombinant plasmid, the vector genome does not include the portion of the "plasmid" that does not correspond to the vector genome array of the recombinant plasmid. This non-vector genome portion of the recombinant plasmid can be referred to as the "plasmid backbone", which is important for plasmid cloning and amplification, and this process is necessary for propagation and recombinant virus production, but is not itself packaged or encapsulated into viral (e.g., AAV) particles. Thus, "vector genome" refers to the nucleic acid packaged or encapsulated by a virus (e.g., AAV).

[0041]

[0045] As used herein, the term "serotype" with respect to an AAV vector means a capsid that is serologically distinguishable from other AAV serotypes. Serological distinctiveness is determined based on the lack of cross-reactivity between antibodies to one AAV compared to another AAV. The difference in cross-reactivity is usually due to differences in the capsid protein sequence / epitope (e.g., differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes).

[0042]

[0046] In the conventional definition, a serotype means that the virus of interest has been tested against sera specific to all existing and characterized serotypes for neutralizing activity, and no antibody that neutralizes the virus of interest has been found. As more naturally occurring virus isolates are discovered and / or capsid variants are generated, there may or may not be serological differences from any of the currently existing serotypes. Thus, if a new virus (e.g., AAV) has no serological differences, this new virus (e.g., AAV) is a subgroup or variant of the corresponding serotype. In many cases, it is still necessary to perform serological testing for neutralizing activity against mutant viruses with capsid sequence modifications to determine whether they are another serotype according to the conventional serotype definition. Thus, for convenience and to avoid repetition, the term "serotype" broadly refers to both serologically distinct viruses (e.g., AAV) and serologically indistinguishable viruses (e.g., AAV) that may be within a subgroup or variant of a given serotype.

[0043]

[0047] The rAAV vector / particle includes any viral strain or serotype. For example, but not limited to, the rAAV vector genome or particle (such as the capsid including VP1, VP2, and / or VP3) can be based on any AAV serotype such as AAV-1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, -rh74, -rh10, or AAV-2i8. Such rAAV vectors / particles can be based on the same strain or serotype (or subgroup or variant), or can be different from each other. For example, but not limited to, the rAAV vector genome or particle (capsid) based on one serotype genome can be identical to one or more capsid proteins that package the vector. Further, the rAAV vector genome can be based on an AAV serotype genome that is different from the one or more capsid proteins that package the vector genome, in which case at least one of the three capsid proteins can be from a different AAV serotype, such as AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV-2i8, SPK1 (SEQ ID NO: 1), SPK2 (SEQ ID NO: 2), or a variant thereof. More specifically, the rAAV2 vector genome can include AAV2 ITRs, but can include capsids from different serotypes such as AAV1, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV-2i8, SPK1 (SEQ ID NO: 1), SPK2 (SEQ ID NO: 2), or a variant thereof. Thus, rAAV vectors include gene / protein sequences identical to those characteristic of a particular serotype, as well as "mixed" serotypes also referred to as "pseudotypes".

[0044]

[0048] In certain embodiments, the rAAV vector comprises, or consists of, a capsid sequence that is at least 70% or more identical (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) to one or more of the AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV-2i8, SPK1 (SEQ ID NO: 1), or SPK2 (SEQ ID NO: 2) capsid proteins (VP1, VP2, and / or VP3 sequences). In certain embodiments, the rAAV vector comprises, or consists of, a sequence that is at least 70% or more identical (e.g., 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, etc.) to one or more of the AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74 or -rh10 ITR(s).

[0045]

[0049] In certain embodiments, the rAAV vector / particle includes AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, and AAV-2i8 variants (e.g., ITR and capsid variants such as amino acid insertions, additions, substitutions, and deletions), for example, those described in International Publication No. WO2013 / 158879 (International Application PCT / US2013 / 037170), International Publication No. WO2015 / 013313 (International Application PCT / US2014 / 047670), and U.S. Patent Application Publication No. 2013 / 0059732 (U.S. Application No. 13 / 594,773).

[0046]

[0050] AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, -rh74, -rh10, AAV-2i8, SPK1 (SEQ ID NO: 1), SPK2 (SEQ ID NO: 2), and rAAV particles such as variants, hybrids, and chimeric sequences can be constructed using recombinant techniques known to those skilled in the art to contain one or more heterologous polynucleotide sequences (transgenes) adjacent to one or more functional AAV ITR sequences at the 5' and / or 3' ends. rAAV vectors typically retain at least one functional adjacent ITR sequence(s) as required for rescue, replication, and packaging of the recombinant vector into rAAV vector particles. Thus, the rAAV vector genome contains in cis the sequences required for replication and packaging (e.g., functional ITR sequences).

[0047]

[0051] Host cells for generating recombinant AAV particles include, but are not limited to, microorganisms, yeast cells, insect cells, and mammalian cells that can be or have been used as recipients of heterologous rAAV vectors. Cells from the stable human cell line HEK293 (readily available, for example, through the American Type Culture Collection under accession number ATCC CRL1573) can be used. In certain embodiments, recombinant AAV particles are produced using a modified human embryonic kidney cell line (e.g., HEK293) transformed with an adenovirus type 5 DNA fragment and expressing the adenovirus E1a and E1b genes. The modified HEK293 cell line is readily transfected and provides a particularly convenient platform for generating rAAV particles. Other host cell lines suitable for recombinant AAV production are described in International Application PCT / 2017 / 024951.

[0048]

[0052] In certain embodiments, the AAV helper function is introduced into host cells by transfecting the host cells with an AAV helper construct either before or simultaneously with transfection of the AAV expression vector. Thus, AAV helper constructs are sometimes used to provide at least transient expression of the AAV rep and / or cap genes to complement the missing AAV functions required for productive AAV transduction. AAV helper constructs often lack AAV ITRs and are unable to replicate or package themselves. These constructs can be in the form of plasmids, phages, transposons, cosmids, viruses, or virions. A number of AAV helper constructs are described, such as the commonly used plasmids pAAV / Ad and pIM29+45 that encode both Rep and Cap expression products. A number of other vectors encoding Rep and / or Cap expression products are known.

[0049]

[0053] Methods for generating recombinant AAV vectors / particles capable of transducing mammalian cells are known in the art. For example, recombinant AAV vectors / particles can be generated as described in U.S. Patent No. 9,408,904; International Applications PCT / US2017 / 025396 and PCT / US2016 / 064414.

[0050]

[0054] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to refer to all forms of nucleic acids, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids include genomic DNA, cDNA and antisense DNA, as well as spliced or unspliced mRNA, rRNA, tRNA and inhibitory DNA or RNA (RNAi, e.g., small or short hairpin (sh) RNA, microRNA (miRNA), small or short interfering (si) RNA, trans-splicing RNA, or antisense RNA). Nucleic acids include naturally occurring, synthetic, and intentionally modified or recombinant polynucleotides (e.g., variant nucleic acids).

[0051]

[0055] Nucleic acids such as vector genomes, cDNA, genomic DNA, RNA, and fragments thereof can be single-stranded, double-stranded, or triple-stranded, linear or circular, and can be of any length. In the study of nucleic acids, the sequence or structure of a particular nucleic acid may be described herein according to the convention of providing the sequence in the 5' to 3' direction.

[0052]

[0056] "Transgene" is used herein to conveniently refer to a heterologous nucleic acid that is intended to be introduced or has been introduced into a cell or organism. Transgenes include any heterologous nucleic acid such as a nucleic acid encoding TPP1.

[0053]

[0057] The terms "transduce" and its grammatical variations refer to the introduction of a molecule such as an rAAV vector into a cell or host organism. The heterologous nucleic acid / transgene may or may not be integrated into the genomic nucleic acid of the recipient cell. The introduced heterologous nucleic acid may also exist only extrachromosomally or transiently in the recipient cell or host organism.

[0054]

[0058] A "transduced cell" is a cell into which a transgene has been introduced. Thus, a "transduced" cell (e.g., a mammalian cell such as a cell or tissue or organ cell) means a genetic change in the cell after, for example, taking up a nucleic acid (e.g., a transgene) into the cell. Thus, a "transduced" cell is a cell into which an exogenous nucleic acid (e.g., a nucleic acid encoding TPP1) has been introduced, or a progeny thereof. The cell(s) can be propagated and the introduced protein can be expressed. With respect to the use and methods of gene therapy, transduced cells can be in a subject such as a mammal, primate, or human.

[0055]

[0059] An "expression control element" refers to a nucleic acid sequence(s) that affects the expression of an operably linked nucleic acid. The expression control elements described herein include promoters and enhancers. A vector sequence, including an AAV vector, can contain one or more "expression control elements". Typically, such elements are included to facilitate transcription of a suitable heterologous polynucleotide and, optionally, translation (e.g., promoter, enhancer, intron splicing signals, maintenance of the correct reading frame of a gene that allows in-frame translation of the mRNA, and stop codons, etc.). Such elements typically act in cis, referred to as "cis-acting" elements, but may also act in trans.

[0056]

[0060] Expression control can be effected at levels such as transcription, translation, splicing, message stability, etc. Typically, expression control elements that regulate transcription are juxtaposed near the 5' end (i.e., "upstream") of the transcribed nucleic acid. Expression control elements can also be located at the 3' end (i.e., "downstream") of the transcribed sequence or within the transcript (e.g., within an intron). Expression control elements can be positioned adjacent to or distant from the transcribed sequence (e.g., 1 - 10, 10 - 25, 25 - 50, 50 - 100, 100 - 500, or more nucleotides from a polynucleotide), even at considerable distances. Nevertheless, due to the length limitations of the AAV vector, expression control elements are typically within 1 - 1000 nucleotides from the transcription start site of the heterologous nucleic acid.

[0057]

[0061] Functionally, the expression of operably linked nucleic acids can be at least partially controlled by elements (e.g., promoters), such that the elements regulate the transcription of the nucleic acids and, optionally, the translation of the transcripts. Specific examples of expression control elements are promoters, which are typically located 5' to the transcribed nucleic acid sequence. A promoter typically increases the amount of expression from an operably linked nucleic acid compared to the amount expressed in the absence of the promoter.

[0058]

[0062] As used herein, an "enhancer" can refer to a sequence that is located adjacent to a heterologous nucleic acid. Enhancer elements are typically located upstream of promoter elements, but can also function and be located downstream or within the sequence. Thus, enhancer elements can be positioned 10 - 50 base pairs, 50 - 100 base pairs, 100 - 200 base pairs, or 200 - 300 base pairs, or more base pairs upstream or downstream of a heterologous nucleic acid sequence. Enhancer elements typically increase the expression of an operably linked nucleic acid compared to the expression effected by a promoter element.

[0059]

[0063] Expression constructs or cassettes can include regulatory elements that serve to drive expression in a particular cell type or tissue type. Expression control elements (e.g., promoters) that are active in a particular tissue or cell type are referred to herein as "tissue-specific expression control elements / promoters". Tissue-specific expression control elements are typically active in a particular cell or tissue (e.g., the liver). Expression control elements are typically active in a particular cell, tissue, or organ because they are recognized by transcription-activating proteins or other regulators of transcription that are specific to a particular cell type, tissue type, or organ type. Such regulatory elements are known to those of skill in the art (see, e.g., Sambrook et al. (1989) and Ausubel et al. (1992)).

[0060]

[0064] The expression control element also includes a ubiquitous or non-discriminatory promoter / enhancer that can drive the expression of a polynucleotide in many different cell types. Such elements include, but are not limited to, the cytomegalovirus (CMV) immediate early promoter / enhancer sequence, the Rous sarcoma virus (RSV) promoter / enhancer sequence, and other viral promoters / enhancers that are active in various mammalian cell types, or synthetic elements that do not occur naturally (see, for example, Boshart et al., Cell, 41:521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmic β-actin promoter, and the phosphoglycerate kinase (PGK) promoter.

[0061]

[0065] Expression control elements can also confer expression in a regulatable manner, i.e., a signal or stimulus can increase or decrease the expression of an operably linked heterologous polynucleotide. A regulatable element that increases the expression of an operably linked polynucleotide in response to a signal or stimulus is also referred to as an "inducible element" (i.e., induced by the signal). Specific examples include, but are not limited to, hormone (e.g., steroid) - inducible promoters. Typically, the amount of increase or decrease conferred by such an element is proportional to the amount of signal or stimulus present, such that the greater the amount of signal or stimulus, the greater the increase or decrease in expression. Regulatable expression control elements include, for example, but are not limited to, zinc - inducible sheep metallothionein (MT) promoter; steroid hormone - inducible mouse mammary tumor virus (MMTV) promoter; T7 polymerase promoter system (International Publication No. WO98 / 10088); tetracycline repression system (Gossen et al., Proc. Natl. Acad. Sci. USA, Vol. 89: 5547 - 5551 (1992)); tetracycline induction system (Gossen et al., Science, Vol. 268: 1766 - 1769 (1995); also see Harvey et al., Curr. Opin. Chem. Biol. 2: 512 - 518 (1998)); RU486 induction system (Wang et al., Nat. Biotech. 15: 239 - 243 (1997) and Wang et al., Gene Ther. 4: 432 - 441 (1997)); and rapamycin induction system (Magari et al., J. Clin. Invest. 100: 2865 - 2872 (1997); Rivera et al., Nat. Medicine. 2: 1028 - 1032 (1996)). Other regulatable control elements that can be used in the present invention are those regulated by specific physiological states, such as temperature, acute phase, development.

[0062]

[0066] The expression control element also includes natural element(s) of a heterologous polynucleotide. Natural control elements (e.g., a promoter) can be used in the present invention when it is desirable for the expression of the heterologous polynucleotide to mimic natural expression. Natural elements can be used in the present invention when the expression of the heterologous polynucleotide is regulated transiently or developmentally, or in a tissue-specific manner, or in response to a specific transcriptional stimulus. Other natural expression control elements such as introns, polyadenylation sites or Kozak consensus sequences can also be used.

[0063]

[0067] The term "operably linked" means that the regulatory sequences necessary for the expression of a nucleic acid sequence are placed in an appropriate position relative to the sequence so as to effect the expression of the nucleic acid sequence. This same definition may apply to the sequences of nucleic acid sequences and transcriptional control elements (e.g., promoters, enhancers, and termination elements) in an expression vector, such as an rAAV vector.

[0064]

[0068] In the example of an expression control element operably linked to a nucleic acid, the relationship is such that the control element regulates the expression of the nucleic acid. More specifically, for example, but not limited to, two DNA sequences that are operably linked are arranged (cis or trans) in such a relationship that at least one DNA sequence can exert a physiological effect on the other sequence.

[0065]

[0069] Thus, additional elements for the vector include, but are not limited to, one or more copies of the AAV ITR sequence, or expression control (e.g., promoter / enhancer) elements adjacent to a sequence such as an intron, a transcription termination signal or stop codon, 5' or 3' untranslated regions (e.g., a polyadenylation (polyA) sequence).

[0066]

[0070] Additional elements include, for example, fillers or stuffer polynucleotide sequences to improve packaging and reduce the presence of contaminating nucleic acids. AAV vectors typically accept DNA inserts having a size range generally from about 4 kb to about 5.2 kb, or slightly larger. Thus, for shorter sequences, a stuffer or filler is included to adjust the length to approximately or the normal size of the viral genomic sequence that is acceptable for packaging the AAV vector within the viral particle. In certain embodiments, the filler / stuffer nucleic acid sequence is an untranslated (non-protein coding) segment of nucleic acid. For nucleic acid sequences less than 4.7 kb, the filler or stuffer polynucleotide sequence has a length having an overall length of about 3.0 - 5.5 kb, or about 4.0 - 5.0 kb, or about 4.3 - 4.8 kb when combined with the sequence (e.g., when inserted into a vector).

[0067]

[0071] The term "isolated," when used as a modifier of a composition, means that the composition is made by human hand or is completely or at least partially separated from its natural in vivo environment. Generally, isolated compositions are substantially free of one or more substances with which they are normally associated in nature, such as, without limitation, one or more proteins, nucleic acids, lipids, carbohydrates, cell membranes.

[0068]

[0072] The term "isolated" does not exclude combinations produced by human hand, such as, without limitation, rAAV sequences, or rAAV particles that package or encapsidate an AAV vector genome and a pharmaceutical formulation. The term "isolated" also does not exclude alternative physical forms of a composition, such as hybrid / chimeric, multimeric / oligomeric, modified (e.g., phosphorylated, glycosylated, lipidated), or derivatized forms, or forms expressed in host cells produced by human hand.

[0069]

[0073] The term "substantially pure" refers to a preparation containing at least 50-60% by weight of the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.). The preparation can contain at least 75% by weight, or at least 85% by weight, or about 90-99% by weight of the compound of interest. The purity is measured by a method suitable for the compound of interest (e.g., chromatography, agarose or polyacrylamide gel electrophoresis, HPLC analysis, etc.).

[0070]

[0074] When referring to a specific nucleotide sequence or amino acid sequence, the phrase "consisting essentially of" means a sequence having the characteristics of the given SEQ ID NO. For example, without limitation, when used with reference to an amino acid sequence, this phrase includes the sequence itself, as well as molecular modifications that do not affect the basic and novel properties of the sequence.

[0071]

[0075] Nucleic acids, expression vectors (e.g., AAV vector genomes), and plasmids containing nucleic acids encoding TPP1 can be prepared using recombinant DNA technology methods. The availability of nucleotide sequence information enables the preparation of the isolated nucleic acid molecules of the present invention by various means. Nucleic acids encoding TPP1 can be produced using various standard cloning and recombinant DNA techniques, via cell expression or in vitro translation and chemical synthesis techniques. The purity of the polynucleotide can be determined by, for example, but not limited to, sequencing, gel electrophoresis, etc. For example, but not limited to, nucleic acids can be isolated using hybridization or computer-based database screening techniques. Such techniques include, but are not limited to: (1) hybridizing a genomic DNA or cDNA library with a probe to detect homologous nucleotide sequences; (2) performing antibody screening using, for example, but not limited to, an expression library to detect polypeptides having shared structural features; (3) performing polymerase chain reaction (PCR) on genomic DNA or cDNA using primers capable of annealing to the nucleic acid sequence of interest; (4) computationally searching a database of related sequences; and (5) performing differential screening of a subtracted nucleic acid library.

[0072]

[0076] The nucleic acid can be maintained as DNA in any convenient cloning vector. In certain embodiments, the clone is maintained in a plasmid cloning / expression vector, e.g., pBluescript (Stratagene, La Jolla, CA), which is grown in a suitable Escherichia coli (E. coli) host cell. Alternatively, the nucleic acid can be maintained in a vector suitable for expression in mammalian cells, e.g., but not limited to, an AAV vector. If post-translational modifications affect protein function, the nucleic acid molecule can be expressed in mammalian cells.

[0073]

[0077] In certain embodiments, the rAAV vector may optionally contain regulatory elements necessary for the expression of heterologous nucleic acids in cells arranged in such a way as to permit the expression of the encoded protein in the host cell. Such regulatory elements necessary for expression include, but are not limited to, promoter sequences, enhancer sequences, and transcription start sequences described herein and known to those of skill in the art.

[0074]

[0078] The methods and uses of the present invention include the delivery (transduction) of nucleic acids (transgenes) to host cells, including dividing and / or non-dividing cells. The nucleic acids, rAAV vectors, methods, uses, and pharmaceutical formulations of the present invention are further useful in methods of delivering, administering, or providing a sequence encoded by a heterologous nucleic acid to a subject in need thereof as a therapeutic method. In this way, the nucleic acid is transcribed in vivo in the subject and a protein is produced. The subject can benefit from or require the protein because the subject is deficient in the protein or because the production of the protein in the subject confers some therapeutic effect, either as a therapeutic method or otherwise.

[0075]

[0079] The present invention is useful in animals, including human and veterinary applications. Accordingly, suitable subjects include mammals such as humans, as well as non-human mammals. The term "subject" refers to an animal, typically a mammal such as a human, non-human primate (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), domestic animals (dogs and cats), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetal, neonatal, infant, pediatric, and young adult subjects. Subjects include animal disease models, for example, but not limited to, mice and other animal models of protein / enzyme deficiencies such as CLN2.

[0076]

[0080] Subjects suitable for treatment according to the present invention include subjects having or at risk of having TPP1 deficiency or depletion, or subjects that produce abnormal, partially functional or non-functional TPP1. Subjects can be tested for TPP1 expression and / or activity to determine whether such subjects are suitable for treatment by the methods of the present invention. Subjects can also be tested for mutations in the endogenous nucleic acid encoding TPP1. Certain genetic mutations are known to reduce or disrupt TPP1 activity. Subjects suitable for treatment according to the present invention also include subjects who would benefit from TPP1. Treated subjects can be monitored periodically after treatment, e.g., every 1-4 weeks, every 1-6 months, every 6-12 months, or every 1, 2, 3, 4, 5 years or more.

[0077]

[0081] Assays for detecting and / or measuring TPP1 activity are known in the art and include those described in Liu et al., 2017, Clin. Chem., 63:1118-1126, doi:10.1373 / clinchem.2016.269167, and Barcenas et al., 2014, Anal. Chem., 87:7962-7968.

[0078]

[0082] Subjects can be tested for immune responses, e.g., antibodies to AAV. Thus, candidate subjects can be screened prior to treatment by the methods of the present invention. Subjects can also be tested for antibodies to AAV after treatment and, optionally, monitored for a period of time after treatment. Subjects that generate AAV antibodies can be treated with immunosuppressive agents or other regimens described herein.

[0079]

[0083] In addition, subjects suitable for treatment according to the present invention include subjects having or at risk of generating antibodies against AAV (anti-AAV antibodies). rAAV vectors can be administered or delivered to such subjects using several techniques. For example, but not limited to, AAV empty capsids (i.e., AAV lacking the vector genome) can be delivered to bind to anti-AAV antibodies in the subject, thereby enabling the rAAV vector containing the heterologous nucleic acid to transduce the cells of the subject.

[0080]

[0084] As described herein, rAAV is useful as a gene therapy vector because it can penetrate cells and introduce nucleic acid / genetic material into cells. Since AAV is not associated with human pathogenic diseases, rAAV vectors can deliver heterologous polynucleotide sequences (e.g., therapeutic proteins and agents) to human patients without causing substantial AAV etiology or disease.

[0081]

[0085] rAAV vectors have many desirable features for such uses, including tropism for both dividing and non-dividing cells. Initial clinical experience with these vectors has also shown no persistent toxicity, and the immune response is generally minimal or undetectable. AAV is known to infect a wide range of cell types in vivo by receptor-mediated endocytosis or transcytosis. These vector systems have been tested in humans targeting many tissues such as the central nervous system, brain, retinal epithelium, liver, skeletal muscle, airway, joints, and hematopoietic stem cells.

[0082]

[0086] For example, but not limited to, it may be desirable to introduce an rAAV vector that can provide multiple copies of TPP1 and thus a greater amount of TPP1 protein. Improved rAAV vectors and methods for generating these vectors are described in detail in numerous references, patents, and patent applications such as Wright J.F. (Hum Gene Ther 20:698-706, 2009).

[0083]

[0087] The rAAV vector can be administered to a patient, for example, but not limited to, via intracranial injection or via injection in a biocompatible carrier. The rAAV vector can be administered alone or in combination with other molecules. Thus, the rAAV vector and other compositions, agents, drugs, biological agents (proteins) can be incorporated into a pharmaceutical composition. Such pharmaceutical compositions are particularly useful for in vivo or ex vivo administration and delivery to a subject.

[0084]

[0088] In certain embodiments, the pharmaceutical composition also includes a pharmaceutically or biologically acceptable carrier or excipient. Such excipients include any pharmaceutical substance that, by itself, does not induce a harmful immune response in the individual receiving the composition and can be administered without undue toxicity.

[0085]

[0089] As used herein, the terms “pharmaceutically acceptable” and “physiologically acceptable” mean a biologically acceptable formulation, gas, liquid or solid, or a mixture thereof, suitable for one or more routes of administration, in vivo delivery or contact. A “pharmaceutically acceptable” or “physiologically acceptable” composition is a substance that is biological or otherwise undesirable, and can be administered to a subject, for example, without causing a substantial undesirable biological effect. Thus, such pharmaceutical compositions can be used in the present invention when administering, for example, nucleic acids, vectors, virus particles or proteins to a subject.

[0086]

[0090] Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, sugars and ethanol. Pharmaceutically acceptable salts can also be included herein, for example, but not limited to, inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate; and salts of organic acids such as acetate, propionate, malonate, benzoate. In addition, auxiliary substances such as wetting or emulsifying agents, pH buffering substances can be present in such vehicles.

[0087]

[0091] The pharmaceutical composition can be provided as a salt and can be formed with a number of acids including, but not limited to, hydrochloric acid, sulfuric acid, acetic acid, lactic acid, tartaric acid, malic acid, succinic acid, etc. The salt tends to be more soluble in aqueous or other protic solvents than the corresponding free base form. In other cases, the preparation can be a lyophilized powder that, prior to use, in combination with a buffer, in the range of pH 4.5 - 5.5, can contain any or all of the following: 1 - 50 mM histidine, 0.1 - 2% sucrose, and 2 - 7% mannitol.

[0088]

[0092] The pharmaceutical composition can be formulated to be compatible with a particular route of administration or delivery, as described herein or as known to those skilled in the art. Accordingly, the pharmaceutical composition includes carriers, diluents, or excipients suitable for administration by various routes.

[0089]

[0093] Compositions suitable for parenteral administration include aqueous and non - aqueous solutions, suspensions, or emulsions of the active compound, and these preparations are typically sterile and can be isotonic with the blood of the intended recipient. The composition can include, for example, but not limited to, water, buffered saline, Hank's solution, Ringer's solution, dextrose, fructose, ethanol, animal oil, vegetable oil, or synthetic oil. Aqueous injection suspensions can include substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.

[0090]

[0094] Furthermore, suspensions of the active compound can be prepared as suitable oil - injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension can also contain suitable stabilizers or reagents that increase the solubility of the compound to enable the preparation of high - concentration solutions.

[0091]

[0095] Cosolvents and adjuvants can be added to the formulation. Cosolvents include hydroxyl groups or other polar groups, such as, but not limited to, alcohols such as isopropyl alcohol; glycols such as propylene glycol, polyethylene glycol, polypropylene glycol, glycol ethers; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acid esters. Adjuvants can include, for example, but not limited to, surfactants such as soy lecithin and oleic acid; sorbitan esters such as sorbitan trioleate; and polyvinylpyrrolidone.

[0092]

[0096] After the pharmaceutical compositions are prepared, they can be placed in suitable containers and labeled for treatment. Such labels can include dosage, frequency, and method.

[0093]

[0097] Compositions, methods, and pharmaceutical compositions and delivery systems suitable for use in the present invention are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy (2003), 20th Edition, Mack Publishing Co., Easton, PA; Remington’s Pharmaceutical Sciences (1990), 18th Edition, Mack Publishing Co., Easton, PA; The Merck Index (1996), 12th Edition, Merck Publishing Group, Whitehouse, NJ; Pharmaceutical Principles of Solid Dosage Forms (1993), Technomic Publishing Co., Inc., Lancaster, Pa.; Ansel and Stoklosa, Pharmaceutical Calculations (2001), 11th Edition, Lippincott Williams & Wilkins, Baltimore, MD; and Poznansky et al., Drug Delivery Systems (1980), edited by R.L. Juliano, Oxford, N.Y., pp. 253-315).

[0094]

[0098] "Effective amount" or "sufficient amount" means, in a single or multiple dose, alone or in combination with one or more other compositions (therapeutic agents such as drugs or immunosuppressive agents), treatments, protocols, or therapeutic regimens, a detectable response for any duration (long-term or short-term), any measurable or detectable degree in a subject, or an expected or desirable result for any duration (e.g., minutes, hours, days, months, years, or cure).

[0095]

[0099] The dosage can vary and may depend on the type of disease for which treatment is indicated, its onset, progression, severity, frequency, duration, or probability, the desired clinical endpoint, previous or concurrent treatments, the general health, age, sex, race, or immunological capacity of the subject, and other factors recognized by those skilled in the art. The dosage, number, frequency, or duration can be proportionally increased or decreased as indicated by the side effects, complications, or other risk factors of the treatment or therapy, and the condition of the subject. Those skilled in the art understand the factors that can affect the dosage and timing necessary to provide an amount sufficient to provide a therapeutic or prophylactic benefit.

[0096]

[0100] The dosage to achieve a therapeutic effect, e.g., the dosage of vector genome per kilogram of body weight (vg / kg) of the subject or patient, or the dosage of vector genome per brain of the subject or patient (vg / brain), or the dosage of vector genome delivered to the CNS of the subject or patient (vg / CNS), varies based on several factors including, but not limited to, the route of administration, the level of heterologous polynucleotide expression necessary to achieve the therapeutic effect, the specific disease being treated, any host immune response to the viral vector, the host immune response to the heterologous polynucleotide or expression product (protein), and the stability of the protein being expressed.

[0097]

[0101] Generally, the dosage is greater than about 1.5×10 13 recombinant AAV vector genomes. For example, a dosage of about 5×10 13 recombinant AAV vector genomes or greater than about 5×10 13 ; a dosage of about 1×10 14 recombinant AAV vector genomes or greater than about 1×10 14 ; a dosage of about 5×10 14 recombinant AAV vector genomes or greater than about 5×10 14 ; a dosage of about 1×10 15 recombinant AAV vector genomes or greater than about 1×10 15 ; and a dosage of about 5×10 15Recombinant AAV vector genome or a dose of recombinant AV vector genome greater than about 5×10 15 In certain embodiments, the recombinant AAV vector genome is in the range of about 1.5×10

[0098]

[0102] ~ about 5×10 13 ~ about 5×10 15 In the range of doses of recombinant AAV vector genome; about 1×10 14 ~ about 3×10 15 In the range of doses of recombinant AAV vector genome; about 2×10 14 ~ about 2×10 15 In the range of doses of recombinant AAV vector genome; about 2.5×10 14 ~ about 7.5×10 14 In the range of doses of recombinant AAV vector genome; about 5×10 14 ~ about 5×10 15 In the range of doses of recombinant AAV vector genome; and about 1×10 15 ~ about 5×10 15 In the range of doses of recombinant AV vector genome.

[0099]

[0103] In certain embodiments, the rAAV vector genome is administered at a dose of about 1×10 14 vector genomes, at a dose of about 2×10 14 vector genomes, at a dose of about 3×10 14 vector genomes, at a dose of about 4×10 14 vector genomes, at a dose of about 5×10 14 vector genomes, at a dose of about 6×10 14 vector genomes, at a dose of about 7×10 14 vector genomes, at a dose of about 8×10 14 vector genomes, at a dose of about 9×10 14 vector genomes, at a dose of about 1×10 15 vector genomes, at a dose of about 2×10 15 vector genomes, at a dose of about 3×10 15 vector genomes, at a dose of about 4×10 15 vector genomes, or at a dose of about 5×1015 It is administered at a dose of the vector genome.

[0100]

[0104] In certain embodiments, the dose is about 1.5×10 13 rAAV vg per subject or patient exceeds that of the brain. For example, about 5×10 13 rAAV vg per brain or a dose exceeding about 5×10 13 rAAV vg per brain; about 1×10 14 rAAV vg per brain or a dose exceeding about 1×10 14 rAAV vg per brain; about 5×10 14 rAAV vg per brain or a dose exceeding about 5×10 14 rAAV vg per brain; about 1×10 15 rAAV vg per brain or a dose exceeding about 1×10 15 rAAV vg per brain; and about 5×10 15 rAAV vg per brain or a dose exceeding about 5×10 15 rAAV vg per brain.

[0101]

[0105] In certain embodiments, the rAAV vg is about 1.5×10 13 ~ about 5×10 15 rAAV vg per brain dose range; about 1×10 14 ~ about 3×10 15 rAAV vg per brain dose range; about 2×10 14 ~ about 2×10 15 rAAV vg per brain dose range; about 2.5×10 14 ~ about 7.5×10 14 rAAV vg per brain dose range; about 5×10 14 ~ about 5×10 15 rAAV vg per brain dose range; and about 1×10 15 ~ about 5×10 15 It is administered within the rAAV vg per brain dose range.

[0102]

[0106] In certain embodiments, the rAAV vg is administered at a dose of about 1×10 14 rAAV vg per brain, and at a dose of about 2×10 14 rAAV vg per brain, and at a dose of about 3×10 14Administered at a dose of rAAV vg / brain, approximately 4×10 14 Administered at a dose of rAAV vg / brain, approximately 5×10 14 Administered at a dose of rAAV vg / brain, approximately 6×10 14 Administered at a dose of rAAV vg / brain, approximately 7×10 14 Administered at a dose of rAAV vg / brain, approximately 8×10 14 Administered at a dose of rAAV vg / brain, approximately 9×10 14 Administered at a dose of rAAV vg / brain, approximately 1×10 15 Administered at a dose of rAAV vg / brain, approximately 2×10 15 Administered at a dose of rAAV vg / brain, approximately 3×10 15 Administered at a dose of rAAV vg / brain, approximately 4×10 15 Administered at a dose of rAAV vg / brain, or approximately 5×10 15 Administered at a dose of rAAV vg / brain.

[0103]

[0107] As used herein, "unit dosage form" refers to physically discrete units suitable as a single dosage for a subject to be treated; each unit contains a predetermined quantity, optionally associated with a pharmaceutical carrier (excipient, diluent, vehicle or filler), calculated to produce the desired effect (e.g., prophylactic or therapeutic effect) when administered in one or more doses. The unit dosage form can be, for example, in ampoules and vials containing a liquid composition, or a composition in freeze-dried or lyophilized state; for example, a sterile liquid carrier can be added before in vivo administration or delivery. Individual unit dosage forms can be included in multiple-dose kits or containers. rAAV particles, and pharmaceutical compositions thereof, can be packaged in single or multiple unit dosage forms for ease of administration and uniformity of dosage.

[0104]

[0108] An "effective amount" or "sufficient amount" of a dosage for treatment (e.g., for improvement or to provide a therapeutic benefit or amelioration) is typically effective to provide a response to one, several, or all of the deleterious symptoms, outcomes, or complications of a disease, e.g., to a measurable extent, to one or more deleterious symptoms, disorders, diseases, conditions, or complications caused by or associated with the disease, but it is a satisfactory result to reduce, lower, inhibit, suppress, limit, or control the progression or worsening of the disease.

[0105]

[0109] An effective amount or sufficient amount may be provided in a single administration, but not necessarily so, and may require multiple administrations and may be administered alone or in combination with another composition (e.g., an agent), treatment, protocol, or treatment regimen, but not necessarily so. For example, the amount may be increased proportionally as indicated by the needs of the subject, the type, condition, and severity of the disease being treated, or the side effects (if any) of the treatment. Further, an effective amount or sufficient amount need not be effective or sufficient when administered in a single or multiple dosages without accompanying a second composition (e.g., another drug or agent), treatment, protocol, or treatment regimen. This is because additional dosages, amounts or periods above or exceeding such dosages, or additional compositions (e.g., drugs or agents), treatments, protocols, or treatment regimens can be included for being considered effective or sufficient in a given subject. An amount considered effective also includes an amount that results in a reduction in the use of another treatment, treatment regimen, or protocol, such as the administration of a nucleic acid encoding TPP1 for the treatment of TPP1 deficiency (e.g., CLN2).

[0106]

[0110] Accordingly, the methods and uses of the present invention also include, inter alia, methods and uses that result in a reduced need or use of another compound, agent, drug, therapy regimen, treatment protocol, process, or remedy. Accordingly, according to the present invention, methods and uses are provided that reduce the need or use of another treatment or therapy.

[0107]

[0111] An effective amount or sufficient amount need not be effective in each and every subject treated, or in a majority of the subjects treated in a given group or population. An effective amount or sufficient amount means effectiveness or sufficiency in a particular subject, not in a group or general population. As is typical with such methods, some subjects will show a greater response, or a lesser response or no response, to a given treatment method or use.

[0108]

[0112] Administration or in vivo delivery to a subject can be carried out prior to the onset of adverse symptoms, conditions, complications, etc. caused by or associated with a disease. For example, screening (e.g., genetic) can be used to identify such subjects as candidates for the compositions, methods, and uses of the present invention. Thus, such subjects include those that screen positive for an insufficient amount or lack of a functional gene product (e.g., TPP1 deficiency), or those that produce an abnormal, partially functional, or non-functional gene product (e.g., TPP1).

[0109]

[0113] Administration or in vivo delivery to a subject according to the methods and uses of the present invention disclosed herein can be carried out within 1 to 2 hours, 2 to 4 hours, 4 to 12 hours, 12 to 24 hours, or 24 to 72 hours after the subject has a disease that is the target of treatment, has one or more symptoms of the disease, or has been screened and identified as positive as described herein, even if the subject does not have one or more symptoms of the disease. Of course, the methods and uses of the present invention can be carried out 1 to 7 days, 7 to 14 days, 14 to 24 days, 24 to 48 days, 48 to 64 days or more, months or years after the subject has a disease that is the target of treatment, has one or more symptoms of the disease, or has been screened and identified as positive as described herein.

[0110]

[0114] The term "improve" means a detectable or measurable improvement in the disease or its symptoms, or the underlying cellular response in question. A detectable or measurable improvement includes a subjective or objective decrease, reduction, inhibition, suppression, limitation or control in the incidence, frequency, severity, progression, or duration of the disease, or a complication resulting from or associated with the disease, or an improvement in the symptoms or underlying cause or consequence of the disease, or a recovery from the disease.

[0111]

[0115] In the case of CLN2, an effective amount is an amount that suppresses, reduces, or improves visual impairment, cognitive developmental disorder or poor cognitive development, loss of motor control or seizures. An effective amount is also an amount that stabilizes, inhibits or prevents worsening of the adverse symptoms of CLN2.

[0112]

[0116] The therapeutic dosage depends, among other factors, on the age and general condition of the subject, and the severity of the disease or disorder. The therapeutically effective amount in humans can be within a relatively wide range that can be determined by a physician based on the response of the individual patient.

[0113]

[0117] Compositions such as pharmaceutical compositions can be delivered to a subject to enable the production of the encoded protein. In certain embodiments, the pharmaceutical composition contains a sufficient amount of genetic material to enable the recipient to produce a therapeutically effective amount of the protein in the subject.

[0114]

[0118] The composition can be formulated and / or administered in any sterile, biocompatible pharmaceutical carrier, including but not limited to saline, buffered saline, dextrose, and water. The composition can be formulated and / or administered to a patient alone or in combination with other agents that affect hemostasis (e.g., cofactors).

[0115]

[0119] The treatment methods of the present invention include systemic, local or topical delivery and administration, or by any route, such as injection or infusion. Delivery of pharmaceutical compositions in vivo can generally be achieved by injection. For example, rAAV vectors / particles can be administered intracranially, for example, within the CNS, particularly to a part of the brain such as the lateral ventricle.

[0116]

[0120] The treatment methods and rAAV vectors according to the present invention include combination therapies that include the additional use of any compound, agent, drug, treatment or other treatment regimen or protocol having a desired therapeutic, beneficial, additive, synergistic or complementary activity or effect. Exemplary combination compositions and treatments include second active agents such as biological agents (proteins), agents (e.g., immunosuppressants) and drugs. Such biological agents (proteins), agents, drugs, treatments and therapies can be administered or implemented prior to, substantially simultaneously with, or subsequent to any other method or treatment according to the present invention.

[0117]

[0121] The compound, agent, drug, treatment or other therapy regimen or protocol can be administered as a combination composition, or separately, such as simultaneously with or sequentially (before or after delivery) with the delivery or administration of the nucleic acid, vector, or rAAV particle. Accordingly, the present invention provides combinations in which the treatment methods according to the present invention are practiced in combination with any compound, agent, drug, therapy regimen, treatment protocol, process, therapeutic agent or composition described herein or known to those skilled in the art. The compound, agent, drug, therapy regimen, treatment protocol, process, remedy or composition can be administered or implemented prior to, substantially simultaneously with, or subsequent to the administration of the nucleic acid, vector or rAAV particle administered to the patient according to the present invention.

[0118]

[0122] In certain embodiments, at least one immunosuppressant is administered to the subject prior to, substantially simultaneously with, or subsequent to administration of the rAAV vector to the subject. In certain embodiments, the immunosuppressant is an anti-inflammatory agent. In certain embodiments, the immunosuppressant is a steroid. In certain embodiments, the immunosuppressant is prednisone, cyclosporine (e.g., cyclosporine A), mycophenolic acid, rituximab or a derivative thereof.

[0119]

[0123] Strategies to reduce (overcome) or avoid humoral immunity against AAV in gene transfer include administration of high vector doses, use of AAV empty capsids as decoys to adsorb anti-AAV antibodies, administration of immunosuppressive drugs to reduce, decrease, inhibit, prevent or eradicate the humoral immune response against AAV, changing the AAV capsid serotype or engineering the AAV capsid to be less sensitive to neutralizing antibodies, use of plasma exchange cycles to adsorb anti-AAV immunoglobulins, thereby reducing the anti-AAV antibody titer, and use of delivery techniques such as balloon catheters followed by saline washes. Such strategies are described in Mingozzi et al., 2013, Blood, 122:23-36.

[0120]

[0124] Exemplary ratios of AAV empty capsids to rAAV vectors can be in the range of about 100:1 to 50:1, about 50:1 to 25:1, about 25:1 to 10:1, about 10:1 to 1:1, about 1:1 to 1:10, about 1:10 to 1:25, about 1:25 to 1:50, or about 1:50 to 1:100 or therebetween. The ratio can also be about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0121]

[0125] The amount of AAV empty capsids to administer can be calibrated based on the amount (titer) of AAV antibodies produced in a particular subject.

[0122]

[0126] AAV antibodies can be pre - existing and can be present at levels that reduce or block transduction of the target cell with the TPP1 gene - transfer vector. Alternatively, AAV antibodies can be expressed after exposure to AAV or administration of an AAV vector. When such antibodies are expressed after administration of an AAV vector, these subjects can be treated accordingly.

[0123]

[0127] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or study of the present invention, suitable methods and materials are described herein.

[0124]

[0128] All patents, patent applications, publications, and other references, GenBank citations, and ATCC citations cited herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0125]

[0129] All features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Thus, unless specifically stated otherwise, the disclosed features (e.g., nucleic acids encoding TPP1, expression cassettes containing nucleic acids encoding TPP1, and rAAV particles containing nucleic acids encoding TPP1) are examples of members of equivalent or similar features.

[0126]

[0130] As used herein, the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a nucleic acid" includes a plurality of such nucleic acids, reference to "a vector" includes a plurality of such vectors, and reference to "a virus" or "a particle" includes a plurality of such viruses / particles.

[0127]

[0131] As used herein, all numerical values or numerical ranges include integers within such ranges and values or fractional parts of integers within the ranges, unless the context clearly indicates otherwise. Thus, for purposes of illustration, reference to 86% or greater identity includes 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, etc., as well as 86.1%, 86.2%, 86.3%, 86.4%, 86.5%, etc., 87.1%, 88.2%, 88.3%, 88.4%, 88.5%, etc.

[0128]

[0132] Reference to an integer with "more than" (greater than) or "less than" includes any number greater than or less than the respective numerical value referred to. Thus, for example, reference to greater than 1.5×10 13 includes reference to 1.6×10 13 、1.7×10 13 、1.8×10 13 、1.9×10 13 、2.0×10 13 、2.1×10 13 、2.2×10 13 、2.3×10 13 、2.4×10 13 、2.5×10 13 、2.6×10 13 、2.7×10 13 、2.8×10 13 、2.9×10 13 、3.0×10 13 、3.1×10 13 、3.2×10 13 and so on.

[0129]

[0133] As used herein, all numerical values or ranges include sub-ranges, as well as the values and fractional parts of integers within such ranges, unless the context clearly indicates otherwise. Thus, for illustrative purposes, references to numerical ranges such as 1 to 10 include 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 2 to 3, 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5, 3 to 6, 3 to 7, 3 to 8, 3 to 9, 3 to 10, etc.; and 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc. Thus, references to the range of 1 to 50 include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. up to 50, as well as 1.1, 1.2, 1.3, 1.4, 1.5, etc., 2.1, 2.2, 2.3, 2.4, 2.5, etc.

[0130]

[0134] References to a series of ranges include ranges that combine the boundary values of the different ranges within the series. Thus, for illustrative purposes of references to a series of ranges, for example, 1 to 10, 10 to 20, 20 to 30, 30 to 40, 40 to 50, 50 to 60, 60 to 75, 75 to 100, 100 to 150, 150 to 200, 200 to 250, 250 to 300, 300 to 400, 400 to 500, 500 to 750, 750 to 850 include ranges such as 1 to 20, 1 to 30, 1 to 40, 1 to 50, 1 to 60, 10 to 30, 10 to 40, 10 to 50, 10 to 60, 10 to 70, 10 to 80, 20 to 40, 20 to 50, 20 to 60, 20 to 70, 20 to 80, 20 to 90, 50 to 75, 50 to 100, 50 to 150, 50 to 200, 50 to 250, 100 to 200, 100 to 250, 100 to 300, 100 to 350, 100 to 400, 100 to 500, 150 to 250, 150 to 300, 150 to 350, 150 to 400, 150 to 450, 150 to 500, etc.

[0131]

[0135] The present invention is generally disclosed herein using affirmative language to describe numerous embodiments of the present invention. The present invention also includes embodiments in which specific subjects, such as substances or materials, method steps and conditions, protocols, or procedures, are excluded in whole or in part. For example, in certain embodiments of the present invention, materials and / or method steps are excluded. Thus, even if the present invention is not generally represented herein with respect to those aspects that are not explicitly excluded in the present invention, it is still disclosed herein.

[0132]

[0136] Numerous embodiments of the present invention are described. Nevertheless, those skilled in the art can make various changes and modifications to the present invention to adapt it to various applications and conditions without departing from the spirit and scope of the present invention. Therefore, the following examples are intended to illustrate, in any way, the scope of the claimed invention, but not to limit it.

Examples

[0133] Example 1

[0137] In this study, non-human primates (female and male) of adult rhesus monkeys (Macaca mulatta) were used. The dosing groups were control (vehicle only); low dose (1.0×10 13 vg / animal); medium dose (5.0×10 13 vg / animal); high dose (2.17×10 14 vg / animal).

[0134]

[0138] Number of animals by dosing group: control (n = 3 at each time point), low dose (N = 3 at each time point), medium dose (n = 3 at each time point), and high dose group (n = 4 at each time point). The time points were 30 days and 90 days.

[0135]

[0139] AAV2-CAG-hTTP1 administration: MRI-guided unilateral delivery to the posterior horn of the lateral ventricle using a spinal needle (22G, 3.5” Quinke BD) (Figure 1; vertical line). A total volume of 4 mL was delivered at (100 μL / min).

[0136]

[0140] Cerebrospinal fluid (CSF) analysis included a TPP1 enzyme activity assay and a human TPP1 protein expression assay (WES Western).

[0137] Example 2

[0141] This example includes a description of data showing short - and long - term expression and activity of human TPP1 in the CNS after intracerebroventricular delivery of AAV2 - CAG - human TPP1.

[0138]

[0142] Human TPP was secreted into the CSF of non - human primates after delivery of an AAV - CAG - human TPP1 (also called AAV - CAG - hTPP1) vector targeting ependymal cells of the lateral ventricles of the CNS. Measurable and sustained expression of human TPP1 was present over a 20 - week time course after AAV vector delivery (Figures 2A, 3A, and 4). Furthermore, TPP1 expression levels at all three AAV vector doses resulted in levels at or above the previously reported K of TPP1 (Vuillemenot, B.R. et al., (2014) Toxicol Appl Pharmacol, 277(1), 49 - 57). This indicates a high likelihood of long - term and sustained cellular uptake in the parenchyma of these animals (Katz, M.L. et al., (2015) Sci Transl Med, 7(313); Tecedor, L. (2018) 16th International Conference on NCL, London, UK). TPP1 activity assays confirmed the functional viability of the expressed TPP1 protein (Figures 2B and 3B). Preliminary analysis of post - mortem tissues from animals receiving AAV2 - CAG - hTPP1 showed no significant pathological changes compared to control animals receiving only diluent. Analysis of tissue uptake of expressed hTPP1 in animals has been performed. 取り込み

[0139]

[0143] ​These studies have demonstrated an effective ependymal-directed gene therapy approach that results in the expression of human TPP1 from ependymal cells of the lateral ventricles for the treatment of late-onset infantile neuronal ceroid lipofuscinosis.

[0140] Example 3 Spk1 VP1 capsid (SEQ ID NO: 1): MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL

[0141] Spk2 VP1 capsid (SEQ ID NO: 2): MAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKTAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMASGGGAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRPL

[0142] CAG promoter sequence (SEQ ID NO: 3):

[0143] TPP1 (SEQ ID NO: 4, human): MGLQACLLGLFALILSGKCSYSPEPDQRRTLPPGWVSLGRADPEEELSLTFALRQQNVERLSELVQAVSDPSSPQYGKYLTLENVADLVRPSPLTLHTVQKWLLAAGAQKCHSVITQDFLTCWLSIRQAELLLPGAEFHHYVGGPTETHVVRSPHPYQLPQALAPHVDFVGGLHRFPPTSSLRQRPEPQVTGTVGLHLGVTPSVIRKRYNLTSQDVGSGTSNNSQACAQFLEQYFHDSDLAQFMRLFGGNFAHQASVARVVGQQGRGRAGIEASLDVQYLMSAGANISTWVYSSPGRHEGQEPFLQWLMLLSNESALPHVHTVSYGDDEDSLSSAYIQRVNTELMKAAARGLTLLFASGDSGAGCWSVSGRHQFRPTFPASSPYVTTVGGTSFQEPFLITNEIVDYISGGGFSNVFPRPSYQEEAVTKFLSSSPHLPPSSYFNASGRAYPDVAALSDGYWVVSNRVPIPWVSGTSASTPVFGGILSLINEHRILSGRPPLGFLNPRLYQQHGAGLFDVTRGCHESCLDEEVEGQGFCSGPGWDPVTGWGTPNFPALLKTLLNP

[0144] [Related Applications]

[0001] This patent application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 800,131, filed on February 1, 2019. The entire contents of the above application, including all text, tables, drawings, and sequences, are incorporated herein by reference.

[0145] Preferred embodiments of the present invention are as follows. [1] A method of treating a primate in need of tripeptidyl peptidase 1 (TPP1), comprising: (a) providing a recombinant adeno-associated virus (AAV) vector comprising a nucleic acid encoding TPP1; (b) Administering a predetermined amount of the recombinant AAV vector to the central nervous system (CNS) of the primate, wherein the TPP1 is expressed in the primate, and the step A method comprising. [2] The method according to [1], wherein the primate is a human. [3] The method according to [2], wherein the human has late-onset neuronal ceroid lipofuscinosis (CLN2). [4] The method according to [2], wherein the human is about 1 to 10 years old or older than 10 years old. [5] The method according to [2], wherein the human is about 2 to 5 years old. [6] The method according to any one of [1] to [5], wherein the administration is to the lateral ventricle or the cisterna magna. [7] The method according to [6], wherein the administration is to the occipital horn of the lateral ventricle. [8] The method according to any one of [1] to [7], wherein the recombinant AAV vector is administered unilaterally to one lateral ventricle. [9] The method according to any one of [1] to [7], wherein the recombinant AAV vector is administered bilaterally to each lateral ventricle.

[10] The method according to any one of [1] to [7], wherein the recombinant AAV vector is administered unilaterally or bilaterally multiple times to one or both lateral ventricles.

[11] The method according to any one of [1] to

[10] , wherein the TPP1 is expressed at an increased level in the CNS.

[12] The method according to any one of [1] to

[11] , wherein the TPP1 is expressed or delivered throughout the CNS.

[13] The method according to any one of [1] to

[12] , wherein the TPP1 is expressed in or delivered to ependymal cells.

[14] The method according to any one of [1] to

[13] , wherein the TPP1 is substantially delivered.

[15] The method according to any one of [1] to

[14] , wherein the TPP1 expression is maintained at a level equal to or exceeding that required for maximal uptake of TPP1 by neurons.

[16] The TPP1 expression is K 取り込みmaintained at a level equal to or exceeding K 取り込み wherein K is at least about 60 ng / mL, the method according to any one of [1] to

[14] .

[17] wherein said TPP1 expression is maintained at a level equal to or exceeding K 取り込み maintained at a level equal to or exceeding K 取り込み wherein K is at least about 60 ng / mL to 120 ng / mL, the method according to any one of [1] to

[14] .

[18] wherein said TPP1 expression is maintained at a level exceeding about 120 ng / mL, the method according to any one of [1] to

[14] .

[19] wherein said TPP1 expression is maintained at a level exceeding about 150 ng / mL, or exceeding about 200 ng / mL, or exceeding about 250 ng / mL, or exceeding about 300 ng / mL, the method according to any one of [1] to

[14] .

[20] wherein TPP1 expression is maintained in the CNS for at least about 5 weeks, or at least about 10 weeks, or at least about 20 weeks, the method according to any one of [1] to

[19] .

[21] wherein detectable TPP1 expression or TPP1 activity is maintained in the CNS for at least 5 weeks, or at least 10 weeks, or at least 20 weeks, the method according to any one of [1] to

[19] .

[22] wherein said recombinant AAV vector is at a dose exceeding about 1.5×10 13 AAV vector genomes; about 5×10 13 AAV vector genomes, or at a dose exceeding about 5×10 13 AAV vector genomes; about 1×10 14 AAV vector genomes, or at a dose exceeding about 1×10 14 AAV vector genomes; about 5×10 14 AAV vector genomes, or at a dose exceeding about 5×10 14 AAV vector genomes; about 1×10 15 AAV vector genomes, or at a dose exceeding about 1×10 15 AAV vector genomes; or at a dose exceeding about 5×10 15 AAV vector genomes, or at a dose exceeding about 5×10 15The method according to any one of [1] to

[21] , administered to the above-mentioned CNS at a dose exceeding the AAV vector genome.

[23] The above recombinant AAV vector is about 1.5 × 10 13 to about 5 × 10 15 in the dose range of vector genomes; about 1 × 10 14 to about 3 × 10 15 in the dose range of vector genomes; about 2 × 10 14 to about 2 × 10 15 in the dose range of vector genomes; about 2.5 × 10 14 to about 7.5 × 10 14 in the dose range of vector genomes; about 5 × 10 14 to about 5 × 10 15 in the dose range of vector genomes; or about 1 × 10 15 to about 5 × 10 15 in the dose range of vector genomes, the method according to any one of [1] to

[22] , administered to the above-mentioned CNS.

[24] The above recombinant AAV vector is about 1 × 10 14 at the dose of vector genomes, about 2 × 10 14 at the dose of vector genomes, about 3 × 10 14 at the dose of vector genomes, about 4 × 10 14 at the dose of vector genomes, about 5 × 10 14 at the dose of vector genomes, about 6 × 10 14 at the dose of vector genomes, about 7 × 10 14 at the dose of vector genomes, about 8 × 10 14 at the dose of vector genomes, about 9 × 10 14 at the dose of vector genomes, about 1 × 10 15 at the dose of vector genomes, about 2 × 10 15 at the dose of vector genomes, about 3 × 10 15 at the dose of vector genomes, about 4 × 10 15 at the dose of vector genomes, or about 5 × 10 15 at the dose of vector genomes, the method according to any one of [1] to

[22] , administered to the above-mentioned CNS.

[25] The method according to any one of [3] to

[24] , wherein the method reduces, decreases, or inhibits one or more symptoms of CLN2; or prevents or reduces the progression or worsening of one or more symptoms of CLN2; or stabilizes one or more symptoms of CLN2; or improves one or more symptoms of CLN2.

[26] The method according to

[25] , wherein the one or more symptoms are selected from the group consisting of visual impairment, cognitive developmental disorder or poor cognitive development, loss of motor control, and seizures.

[27] The method according to any one of [1] to

[26] , wherein the nucleic acid encoding TPP1 comprises an expression cassette operably linked to an expression control element.

[28] The method according to

[27] , wherein the expression control element is located 5' to the nucleic acid.

[29] The method according to

[27] or

[28] , wherein the expression control element comprises a CAG (SEQ ID NO: 3) promoter, a cytomegalovirus (CMV) immediate early promoter / enhancer, a Rous sarcoma virus (RSV) promoter / enhancer, an SV40 promoter, a dihydrofolate reductase (DHFR) promoter, or a chicken β-actin (CBA) promoter.

[30] The method according to any one of [1] to

[29] , wherein the heterologous nucleic acid is located between one or more 5' and / or 3' AAV inverted terminal repeats (ITRs).

[31] The method according to

[30] , wherein the one or more AAV ITRs comprise mutated, modified, or variant AAV ITRs that are not processed by the AAV Rep protein.

[32] The method according to

[30] , wherein the one or more AAV ITRs comprise mutated, modified, or variant AAV ITRs that enable or facilitate the formation of a self-complementary reporter transgene genome in the double-stranded inverted repeat sequence structure in the recombinant AAV vector.

[33] The method according to

[32] , wherein the mutated, modified, or variant AAV ITR has a deleted D sequence and / or a mutated, modified, or variant terminal resolution site (TRS) sequence.

[34] The method according to any one of

[30] to

[33] , wherein the recombinant AAV vector comprises, in the 5'→3' direction, a first AAV ITR; a promoter operable in mammalian cells; a heterologous nucleic acid; a polyadenylation signal; and optionally, a second AAV ITR.

[35] The method according to any one of

[30] to

[33] , wherein the one or more ITRs comprise an AAV serotype AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh74, or Rh10 ITR.

[36] The method according to any one of [1] to

[35] , wherein the recombinant AAV vector comprises a VP1, VP2, and / or VP3 sequence that is 60% or more identical to a VP1, VP2, and / or VP3 sequence of AAV serotype AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh74, Rh10, SPK1 (SEQ ID NO: 1), or SPK2 (SEQ ID NO: 2) VP1, VP2, and / or VP3, or a hybrid or chimeric VP1, VP2, and / or VP3 sequence of any of the foregoing AAV serotypes.

[37] The method according to any one of [1] to

[36] , wherein the recombinant AAV vector comprises a VP1, VP2, and / or VP3 capsid protein having 100% sequence identity to a VP1, VP2, and / or VP3 capsid protein selected from the group consisting of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, Rh10, Rh74, SPK1 (SEQ ID NO: 1), and SPK2 (SEQ ID NO: 2) VP1, VP2, and / or VP3 capsid proteins.

[38] The method according to any one of [1] to

[37] , wherein the recombinant AAV vector further comprises a polyadenylation sequence located 3' of the nucleic acid.

[39] The method according to any one of [1] to

[38] , wherein the nucleic acid encoding TPP1, the expression control element, or the polyadenylation sequence has a reduced CpG compared to the wild-type nucleic acid encoding TPP1, the expression control element, or the polyadenylation sequence.

[40] The method according to

[38] or

[39] , wherein the polyadenylation sequence comprises the bovine growth hormone (bGH) polyadenylation sequence.

[41] The method according to any one of [1] to

[34] , wherein the TPP1 is human and comprises or consists of the sequence shown in SEQ ID NO: 4, or a functional variant or polymorphic form thereof.

[42] The recombinant AAV vector is (a) one or more AAV capsids, and (b) one or more AAV inverted terminal repeats (ITRs), wherein the one or more AAV ITRs are adjacent to the 5' or 3' end of the nucleic acid or the expression cassette, the ITR The method according to any one of [1] to

[41] comprising.

[43] The method according to

[42] , further comprising an intron located 5' or 3' of the one or more ITRs.

[44] The method according to

[42] or

[43] , wherein at least one or more of the one or more ITRs and / or the intron are modified to have a reduced CpG.

[45] The method according to any one of [1] to

[44] , wherein the recombinant AAV vector has a capsid serotype comprising an AAV VP1, VP2, and / or VP3 capsid having 90% or more sequence identity with the VP1, VP2, and / or VP3 sequences of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, AAV-2i8, SPK1 (SEQ ID NO: 1), or SPK2 (SEQ ID NO: 2), or a capsid having 95% or more sequence identity with the VP1, VP2, and / or VP3 sequences of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, Rh10, Rh74, AAV-2i8, SPK1 (SEQ ID NO: 1), or SPK2 (SEQ ID NO: 2), or a capsid having 100% sequence identity with the VP1, VP2, and / or VP3 sequences of AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74, AAV-2i8, SPK1 (SEQ ID NO: 1), or SPK2 (SEQ ID NO: 2).

[46] The method according to any one of

[41] to

[45] , wherein the one or more ITRs comprise one or more ITRs of any one of the AAV1, AAV2, AAV3, AAV3B, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, or Rh74 AAV serotypes, or combinations thereof.

[47] The method according to any one of [1] to

[46] , wherein the recombinant AAV vector is in a pharmaceutical composition comprising a biologically compatible carrier or excipient.

[48] The method according to

[47] , wherein the pharmaceutical composition further comprises an empty AAV capsid.

[49] The method according to

[48] , wherein the ratio of the empty AAV capsid to the recombinant AAV vector is within or between about 100:1 to 50:1, about 50:1 to 25:1, about 25:1 to 10:1, about 10:1 to 1:1, about 1:1 to 1:10, about 1:10 to 1:25, about 1:25 to 1:50, or about 1:50 to 1:100. The method according to

[48] , wherein the ratio of the empty AAV capsid to the recombinant AAV vector is about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. The method according to any one of

[47] to

[50] , wherein the pharmaceutical composition further comprises a surfactant.

Claims

[Claim 1] The invention described herein and in the drawings.

Images