Means and methods for AAV gene therapy in humans

Abstract

We provide AAV-based gene therapy vectors for humans. [Solution] The present invention provides an AAV5 gene therapy vector for use in the medical treatment of human patients, wherein the human is subjected to pre-screening using an assay to determine anti-AAV5 antibodies, the human has not been subjected to medical treatment with the AAV5 gene therapy vector prior to the medical treatment, the human has an anti-AAV5 antibody level corresponding to at most the 95th percentile of anti-AAV5 antibody levels observed in the human population, and the human has shown a positive test result for anti-AAV5 antibodies. The present invention also provides a method for determining whether a human patient is eligible to receive medical treatment with the AAV5 gene therapy vector.

Description

[Technical Field]

[0001] The present invention relates to means and methods for AAV-based gene therapy in humans. In particular, the present invention relates to the treatment of human patients who may be suspected of having antibodies against AAV intended for therapeutic use. [Background technology]

[0002] Adeno-associated virus (AAV) is considered one of the most promising viral vectors for human gene therapy. AAV has the ability to efficiently infect both dividing and non-dividing human cells. The wild-type AAV virus genome is integrated into a single chromosomal region in the host cell genome, and most importantly, even though AAV is present in many humans, it is not associated with any disease. Given these advantages, recombinant adeno-associated virus (rAAV) is being evaluated in gene therapy clinical trials for hemophilia B, malignant melanoma, cystic fibrosis, and other diseases. Numerous clinical trials and approvals of gene therapy drugs in Europe, such as Alipogene tiparvovec (Glybera®, uniQure), suggest that AAV has the potential to become a main stay in clinical practice.

[0003] One major challenge to the successful administration of AAV vectors is overcoming the presence of neutralizing antibodies (immunoglobulins) (NAbs) that develop after exposure to wild-type AAV or AAV-based vectors. In either case, serotype-specific neutralizing antibodies against the viral capsid protein can reduce the efficiency of gene transfer using the same serotype of AAV.

[0004] Compared to other serotypes, relatively low endemic NAB titers have been observed for the AAV5 serotype in humans (Boutin et al., Hum Gene Ther 2010, 21:704-712). In human treatment with AAV, such low endemic NAB titers have already been reported to affect transduction, resulting in severely reduced transgene expression (Manno et al., Nature Medicine, 2006, 12(3), 342-347). Therefore, the general consensus in this field is to completely avoid treating patients with high NAB titers. Thus, current clinical practice for existing immunology involves screening human patients for exclusion if a patient has neutralizing antibodies against the AAV capsid (Brimble et al., Expert Opin Biol Ther 2016, 16(1):79-92, and Boutin et al., Hum Gene Ther 2010, 21:704-712). Immunosuppressive regimens have been tested to reduce the formation of NAbs as soon as the first dose is administered, enabling a second dose (Corti et al., Mol Ther-Meth Clin Dev (2014) 1, 14033; Mingozzi et al., Mol Ther, Vol. 20, No. 7, 1410-1416; McIntosh et al., Gene Ther 2012, 19, 78-85). Furthermore, strategies including plasmapheresis and the use of immunosuppressive regimens have been suggested to overcome existing antibodies (e.g., Chicoine et al., Mol Ther 2014, Vol. 22, No. 2, 338-347; Hurlbut et al., Mol Ther 2010, Vol. 18, No. 11, 1983-1984, and Mingozzi et al., Mol Ther, Vol. 20, No. 7, 1410-1416). These strategies have been tested in animal models and have shown limited success.

[0005] Therefore, there is a need in the art to enable the administration of rAAV gene therapy vectors to human patients who have or are suspected of having AAV neutralizing antibodies. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] The inventors have now surprisingly found, particularly with respect to AAV5 gene therapy vectors, that human patients with endemic existing anti-AAV5 antibodies (i.e., existing anti-AAV5 antibodies resulting from endemic exposure) may be considered eligible for treatment, contrary to the suggestions of the current art. This finding is in contrast to the opinion in the prior art that the presence of neutralizing antibodies should be considered an exclusion criterion for patients participating in clinical trials, for example. In other words, the opinion in the current art is that patients with antibodies against AAV5, and more particularly endemic existing antibodies against AAV5, are considered ineligible for treatment with AAV5 gene therapy vectors. The remarkable finding disclosed herein that human patients with endemic, pre-existing anti-AAV5 antibodies may be considered eligible for treatment relates not only to a subgroup of human patients found to have, for example, very low levels of pre-existing anti-AAV5 antibodies, but rather to most, if not all, of the human population that scores positive for pre-existing anti-AAV5 antibodies and has never been subjected to any AAV5 gene therapy.

[0007] Accordingly, in one embodiment, the present invention provides an AAV5 gene therapy vector for use in the medical treatment of a human patient, wherein the human patient has not been subjected to pre-screening using an assay to determine anti-AAV5 antibodies, and the human has not been subjected to medical treatment with the AAV5 gene therapy vector prior to the medical treatment. In other words, the present invention provides an AAV5 gene therapy vector for use in the medical treatment of a human, wherein the anti-AAV5 antibody status is unknown, and the human has not been subjected to medical treatment with the AAV5 gene therapy vector prior to the medical treatment. According to one embodiment, the present invention may enable the treatment of a patient who has not been treated with the AAV5 gene therapy vector prior to, or prior to, screening for the presence of anti-AAV5 antibodies, or the inclusion of the patient in a clinical trial.

[0008] In a further embodiment, an AAV5 gene therapy vector for use in human medical treatment is provided, wherein the human is subjected to pre-screening using an assay for determining anti-AAV5 antibodies, the human has not been subjected to medical treatment with the AAV5 gene therapy vector prior to the medical treatment, and the human has an anti-AAV5 antibody level that corresponds to at most the 100th percentile, preferably at most the 95th percentile, of the anti-AAV5 antibody levels observed in human populations. The human patient being tested is preferably positive for anti-AAV5 antibodies. [Brief explanation of the drawing]

[0009] [Figure 1A] This shows the NAb assay results for pre-treatment samples. A: This is a graph of the neutralization results for 10 pre-treatment samples. The 50 percent mark is indicated by a dotted line. [Figure 1B]B: Curve fitting results for the three positive samples 3, 4, and 5. The four-parameter curve was fitted using nonlinear regression. Titer was calculated as the theoretical dilution at which the fitted curve passes the 50% mark (shown above the horizontal axis). [Figure 2A] This is a graph of AAV5 neutralizing antibody versus total anti-AAV5 antibody. It shows the results of neutralizing (NAb) titer versus total (TAb) ELISA reported in a population screening survey. Each open symbol represents a pair of NAb and TAb results for a single healthy individual. NAb and TAb results for treated patients are shown overlaid (▲ (black triangle), indicating designated study subjects 3, 4, and 5). [Figure 2B] This is a graph of AAV5 NAb titer versus FIX level. The percentage of FIX activity in Cohort 1 after drug administration is plotted against the NAb titer before drug administration. [Figure 3] This is a diagram of the AAV NAb titer scale. The percentiles for a human population (50 subjects) are depicted in relation to NAb titer relative to AAV5. Note that the NAb titers observed in the human population are considerably different from those observed in human patients treated with AAV5. [Figure 4] The VP1 amino acid sequence of wild-type AAV5 is depicted. The amino acid start positions of VP2 (T, due to the ACG start site) and VP3 (M) are underlined. [Figure 5] The VP1 amino acid sequence of the hybrid VP1 sequence is depicted, consisting of an N-terminal AAV2-derived VP1 sequence (underlined) linked to AAV5-derived VP2 and VP3 coding sequences. Therefore, the VP1 protein is a hybrid AAV2 / AAV5 capsid protein. The expression construct used for the AAV capsid encoding the hybrid VP1 may also encode the VP2 and VP3 sequences, which are wild-type AAV5 VP2 and VP3 proteins, rather than the hybrid VP2 and VP3 capsid proteins. [Figure 6]The VP1 amino acid sequence of wild-type AAV5 is shown, with an Ala insertion between positions 1 and 2 of the wild-type AAV5 sequence. Thus, the VP1 capsid consists of the AAV5 wild-type sequence with the inserted amino acid, and the encoded VP2 and VP3 proteins are the wild-type AAV5 VP2 and VP3 proteins without modification.

[0010] definition An "AAV vector" refers to a recombinant adeno-associated virus (AAV) vector derived from wild-type AAV by molecular methods. AAV vectors are distinguished from wild-type (wt) AAV vectors in that at least a portion of the viral genome has been replaced with a transgene, which is a nucleic acid that is non-native to the wild-type AAV nucleic acid sequence.

[0011] AAV vectors, comprising a combination of AAV capsid and AAV genome ITR, can be produced using methods known in the art, as described by Pan et al. (J. of Virology (1999) 73:3410~3417), Clark et al. (Human Gene Therapy (1999) 10:1031~1039), Wang et al. (Methods Mol. Biol. (2011) 807:361~404), and Grimm (Methods (2002) 28(2):146~157), which are incorporated herein by reference. Alternatively, AAV vectors can be produced in insect cells using a baculovirus expression system (BEVS). The first baculovirus system for rAAV production was described by Urabe et al. (Urabe et al.

[2002] Human Gene Therapy 13(16):1935-1943), consisting of three baculoviruses, named Bac-Rep, Bac-cap, and Bac-vec. Co-infection of insect cells, such as SF9, with these baculoviruses resulted in the production of rAAV. The properties of such produced rAAV, including its physical and molecular characteristics such as potency, did not differ significantly from rAAV produced in mammalian cells (Urabe

[2002] , cited above). The initial baculovirus system by Urabe (2002, cited above) has been further developed (see, for example, Kohlbrenner et al. (2005) Molecular Therapy 12(6):1217~1225; Urabe et al. (2006) Journal of Virology 80(4):1874~1885; International Publication No. 2007 / 046703; International Publication No. 2007 / 148971; International Publication No. 2009 / 014445, and International Publication No. 2009 / 104964).

[0012] The term "transgene" is used to refer to a nucleic acid that is non-native to the AAV nucleic acid sequence. The term is used to refer to a polynucleotide that can be introduced into a cell or organism. Transgenes include any polynucleotide, such as a gene encoding a polypeptide or protein, a polynucleotide that is transcribed into an inhibitory polynucleotide, or a polynucleotide that is not transcribed (e.g., lacking expression control elements such as a promoter that drives transcription). The transgene is preferably inserted between inverted terminal repeat (ITR) sequences. The transgene may also be an expression construct that includes expression control elements such as a promoter or transcription regulatory sequence operably linked to a coding sequence and a 3' termination sequence.

[0013] "Transduction" refers to the transfer of a transgene into a recipient host cell by a viral vector. Transduction of target cells by the rAAV vector of the present invention leads to the transfer of the transgene contained in the vector into the transduced cells. "Host cell" or "target cell" refers to a cell in which DNA delivery occurs, such as, for example, synoviocytes or synovial cells of an individual. The AAV vector can transduce both dividing and non-dividing cells.

[0014] "Gene" or "coding sequence" refers to a DNA or RNA region that "encodes" a specific protein. The coding sequence is transcribed (DNA) and translated into a polypeptide (RNA) when placed under the control of appropriate regulatory regions such as a promoter. A gene may include several operably linked fragments such as a promoter, 5' leader sequence, intron, coding sequence, and 3' untranslated sequence, and may include a polyadenylation site or signal sequence. A chimeric or recombinant gene is a gene that is not normally found in nature, such as, for example, a gene in which part or all of the transcribed DNA region and the promoter are not naturally associated. "Gene expression" refers to the process by which a gene is transcribed into RNA and / or translated into an active protein.

[0015] Sequence identity and sequence similarity can be determined by the alignment of two peptide or nucleotide sequences using a global or local alignment algorithm, depending on the lengths of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithm (e.g., Needleman-Wunsch) that optimally aligns the sequences over their entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith-Waterman). Subsequently, if the sequences share at least a certain minimum percentage of sequence identity (as defined below) (e.g., optimally aligned by a GAP or BESTFIT program using default parameters), the sequences may be referred to as "substantially identical" or "essentially similar." GAP aligns two sequences over their entire lengths (full length) using the Needleman and Wunsch global alignment algorithms, maximizing the number of matches and minimizing the number of gaps. If the two sequences are of similar length, sequence identity is determined using appropriate global alignment. Generally, default GAP parameters are used that have a gap creation penalty of 50 (nucleotides) / 8 (proteins) and a gap elongation penalty of 3 (nucleotides) / 2 (proteins). For nucleotides, the default score matrix used is nwsgapdna, and for proteins, the default score matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919).Sequence alignment and scores for sequence identity percentage can be determined using computer programs such as the GCG Wisconsin Package, version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open-source software such as the "needle" (using the global Needleman-Wunsch algorithm) or "water" (using the local Smith-Waterman algorithm) programs in EmbossWIN version 2.10.0, using the same parameters as above for GAP or default settings (for both "needle" and "water," and for both protein and DNA alignment, the default gap start penalty is 10.0, the default gap extension penalty is 0.5; the default score matrix is ​​Blosum62 for protein and DNAFull for DNA). Local alignment, such as that using the Smith-Waterman algorithm, is preferred when sequences have substantially different overall lengths. Alternatively, similarity or identity percentages can be determined by searching public databases using algorithms such as FASTA and BLAST.

[0016] As used herein, “gene therapy” means the insertion of a nucleic acid sequence (e.g., a transgene as defined herein) into the cells and / or tissues of an individual for the treatment of a disease. A transgene may be a functional mutant allele that replaces or supplements a defective allele. Gene therapy also includes the insertion of a transgene that inhibits, reduces, or impairs the expression, activity, or function of an endogenous gene or protein, such as an undesirable or abnormal (e.g., pathogenic) gene or protein that is inhibitory in nature. Such a transgene may be exogenous. An exogenous molecule or sequence is understood to be a molecule or sequence that is not normally present in the cells, tissues, and / or individual being treated. Both acquired and congenital diseases are suitable for gene therapy. Therefore, AAV5 gene therapy vector refers to an AAV5 vector for use in gene therapy.

[0017] In this document and in the claims herein, the verb “comprise” and its conjugations are used in the non-restrictive sense of the word, meaning that the items following the word are included, but not items not specifically mentioned are excluded.

[0018] Furthermore, the indefinite article "a" or "an" does not rule out the possibility of more than one element being present, unless the context explicitly requires that there be only one or a single element. Therefore, the indefinite article "a" or "an" usually means "at least one."

[0019] When the words "approximately" or "about" are used in relation to a number (approximately 10, about 10), it is preferable that the value may be 10% greater or less than the given value of 10. [Modes for carrying out the invention]

[0020] As stated, it has been surprisingly found that human patients with endemic, existing anti-AAV5 antibodies may be considered eligible for treatment, particularly with respect to AAV5 gene therapy vectors. This finding contrasts with the common view that the presence of existing anti-AAV antibodies against a particular serotype would make gene therapy treatment using that serotype impossible. Without being constrained by theory, AAV5 may be a serotype in which endemic, existing anti-AAV5 antibody titers are relatively low compared to other serotypes, as anti-AAV5 antibodies are found in the human population. This may be due to differences between serotypes and / or the routes of infection, with or without co-infection with helper viruses. Furthermore, AAV5 is most different from and phylogenetically separated from other primate AAV serotypes, which may also contribute to its relatively low titer. Regardless of what is at the root of the invention, being different from other AAV serotypes makes it possible that most, if not all, people in the human population are eligible for treatment using gene therapy or similar methods based on the AAV5 serotype. These individuals include subsets of the human population that are negative for anti-AAV5 antibodies, and subsets of the human population found to be positive for anti-AAV5 antibodies, but do not include (currently) a very small subset of the human population being treated with AAV5 gene therapy or similar. In the human population being treated with AAV5 gene therapy, high titers of anti-AAV5 antibodies are observed that suggest these individuals are not eligible for treatment with AAV5 gene therapy vectors or similar (approximately 10% compared to individuals endemicly infected with AAV5). 4 (or exceeding that amount).

[0021] Accordingly, in a first embodiment of the present invention, an AAV5 gene therapy vector for use in human medical treatment is provided, wherein the human is not subjected to pre-screening using an assay for determining anti-AAV5 antibodies, and the human has not been subjected to medical treatment using the AAV5 gene therapy vector prior to the medical treatment.

[0022] The complete genomes of AAV5 and other AAV serotypes have been sequenced (Chiorini et al., 1999, J. of Virology, Vol. 73, No. 2, pp. 1309-1319), and the nucleotide sequences are available on GenBank (accession number AF085716; February 23, 2015). Gene therapy vectors based on wild-type AAV5 are understood to contain at least AAV5 capsid proteins, including VP1, VP2, and VP3 capsid proteins that correspond to or are at least substantially identical to the wild-type AAV5 amino acid sequence. Being substantially identical to wild-type AAV5 includes having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity with wild-type AAV5. The AAV5 capsid VP1 protein sequence, which may be subject to sequence identity determination, is shown in Figure 4. Such sequences may be naturally occurring sequences of AAV viruses that deviate from the phylogenetic perspective within the AAV5 clade (as illustrated, for example, in Figure 4).

[0023] A "serotype" is traditionally defined based on the lack of cross-reactivity between antibodies against a single virus compared to another virus. Such differences in cross-reactivity usually result from differences in capsid protein sequences / antigenic determinants (e.g., differences in the VP1, VP2, and / or VP3 sequences of AAV serotypes). Under the traditional definition, a serotype means that the virus of interest has been tested against sera specific to all existing serotypes characterized for neutralizing activity, and no antibodies neutralizing the virus of interest have been found. As more naturally occurring virus isolates are discovered and capsid variants are created, there may or may not be serological differences from any of the currently existing serotypes. For convenience, AAV5 serotypes include AAVs with capsid sequence modifications that are not characterized as distinctly different serotypes, which may also constitute a subgroup or variant of the AAV5 serotype. Such variants generally have substantial sequence identity.

[0024] Non-natural capsid sequences can also be constructed in accordance with the present invention, for example, the amino acid sequence exposed to serum (externally exposed) may be derived from one serotype, while the unexposed amino acid sequence within the capsid may be derived from other serotypes and / or allow for more variations. As the crystal structure of AAV5 is known (e.g., Govindasamy et al., J. Virol. October 2013, Vol. 87, No. 20; 11187-11199), the unexposed and, for example, internal sequences of the AAV5 capsid may be replaced with sequences derived from other serotypes and / or allow for more sequence variations. For example, the VP1 amino acid sequence, which is not contained in VP2 and VP3, is placed internally. This sequence may be derived from serotype 2, for example, while the VP2 and VP3 amino acid sequences may be entirely based on AAV5 (see, for example, Figure 5). Such AAV5 gene therapy vector capsids exhibit serotyping and neutralizing antibody patterns indistinguishable from those of a complete wild-type capsid (see, in particular, International Publication No. 2000028004 and Urabe et al., J Virol, February 2006, Vol. 80, No. 4, pp. 1874-1885). Such non-natural capsid sequences are hybrid sequences, and such hybrid vectors are also understood to be AAV5 gene therapy vectors according to the present invention. Furthermore, AAV5 capsid sequences may also have one or more amino acids inserted or replaced to enhance the manufacture and / or potency of the vector, such as those described in International Publication No. 2015137802 and, for example, those shown in Figure 6. Such slightly modified AAV5 capsids may also be considered to be of the AAV5 serotype.

[0025] The AAV5 vector or AAV5 gene therapy vector according to the present invention is understood to relate to an AAV5 vector capsid containing a vector genome having a gene of interest contained between AAV inverted repeats, which may or may not be AAV5 ITRs. Therefore, the AAV5 vector according to the present invention is a delivery vehicle capable of delivering the vehicle's payload, a vector genome having a transgene, for example, a transgene that would be beneficial to humans, to the vehicle's target cells, for example, liver cells or cardiomyocytes. Thus, the AAV5 vector may be useful in the medical treatment of humans, for example, humans suffering from diseases that may be improved by the delivery of a transgene. As shown in the examples, the transgene may be FIX or its variants, such as the Padua variant, but the transgene is by no means limiting, and further transgenes may be contemplated in the present invention as described herein. In one further embodiment, the human patient may be a male human patient.

[0026] In another embodiment, an AAV5 gene therapy vector is provided for use in the medical treatment of humans, wherein the anti-AAV5 antibody status is unknown (e.g., not determined), and the human has not been subjected to medical treatment with the AAV5 gene therapy vector prior to the medical treatment. As stated, it may not be necessary to test for the presence of antibodies against AAV5 in such patients. As shown in the Examples section, approximately 30% of the human population has been shown to be positive for the presence of TAb or NAb in serum. Since it may not be necessary to test for TAb or NAb in serum, the size of the population eligible for treatment is greatly increased, and furthermore, since it is not necessary to perform NAb or TAb assays before treatment can be initiated, the greatly increased population size makes it easier to treat and select eligible patients. All that may be required is to know whether the human patient has been previously subjected to AAV5 gene therapy treatment. Prior treatment with AAV gene therapy may not be limited to AAV5 only, and may include treatment with other serotypes, e.g., serotype 8. For such subjects, testing may be performed, including NAb or TAb assays, or as described in the Examples section, to confirm that serum NAb or TAb titers remain within the range observed in naive, untreated human patients.

[0027] In another embodiment, an AAV5 gene therapy vector is provided for use in human medical treatment, wherein the human is subjected to pre-screening using an assay to determine anti-AAV5 antibodies, the human has not been subjected to medical treatment with the AAV5 gene therapy vector prior to the medical treatment, and the human has an anti-AAV5 antibody level that corresponds at most to the 95th percentile of anti-AAV5 antibody levels observed in human populations. In this embodiment, human patients who could benefit from gene therapy treatment are pre-screened using an anti-AAV5 antibody assay. As shown in the Examples section, the range of anti-AAV5 antibody levels observed in human populations, i.e., the range of observed anti-AAV5 antibody levels, is about 0 to, or about 1 to about 10,000.

[0028] In this specification, the nth percentile is typically defined as the percentage of the human population that has an anti-AAV5 antibody level determined using a NAb assay or TAb assay, such as those described in the Examples, that falls within the distribution range of 0% to n%. For example, if human patients in a population (who have not been previously treated with an AAV5 vector) have an anti-AAV5 antibody level determined using the NAb assay described in the Examples, it is estimated that this would include up to approximately 100% of the human population, so the total anti-AAV5 antibody level detected in the entire population would be at most 10,000. It may be preferable to treat human patients if they have an anti-AAV5 antibody level at the highest percentile, which corresponds to an anti-AAV5 antibody level of up to 4,500, as determined using the assay described in the Examples. It may be preferable to treat human patients if they have anti-AAV5 antibody levels at the 93rd or 90th percentile, corresponding to NAb levels determined using the assays described in the Examples, at most 3,000 or 1,000, respectively (see Figure 3). According to another embodiment, it may be preferable to treat human patients if they have anti-AAV5 antibody levels at the 99th, 98th, 97th, 96th, 95th, 94th, 93rd, 92nd, 91st, 90th, 80th, or 70th percentile, respectively. Nevertheless, it can be expected that most, if not all, of the population are eligible to receive treatment regardless of anti-AAV5 antibody titer. As shown in the Examples section, any anti-AAV5 antibody assay is sufficient, i.e., either an NAb assay or a TAb assay or similar can be used to determine antibody titers in a human population and determine the 95th, 93rd, or 90th percentile. While the selected human population remains the same, the actual titer values ​​can vary considerably (up to 10,000 for the NAb assay in the example, or up to 5 for the TAb assay). This is because the observed titer values ​​are simply relevant when considering titers holistically within the population.In any case, the anti-AAV5 antibody titer levels determined in the population are understood to be those of at least 50 human individuals as described in the Examples section.

[0029] Accordingly, in a further embodiment, an AAV5 gene therapy vector is provided for use in the medical treatment of a human, wherein the human is subjected to pre-screening using an assay for determining anti-AAV5 antibodies, the human has not been subjected to medical treatment with the AAV5 gene therapy vector prior to the medical treatment, and the human has an anti-AAV5 antibody level, as determined by the NAb ELISA assay described in the Examples, which corresponds at most to the 95th percentile of anti-AAV5 antibody levels observed in the human population. According to another embodiment, it may be preferable to treat a human patient if the anti-AAV5 antibody level is at most the 99th, 98th, 97th, 96th, 95th, 94th, 93rd, 92nd, 91st, 90th, 80th, or 70th percentile.

[0030] In accordance with the present invention, a subpopulation of human populations that would have previously been considered ineligible to receive treatment with AAV5 is now understood to be eligible to receive treatment with an AAV5 gene therapy vector, despite showing a positive test result in an anti-AAV5 antibody assay. Accordingly, in a further embodiment, an AAV5 gene therapy vector for use in the medical treatment of a human is provided, wherein the human has shown a positive test result for an anti-AAV5 antibody and has not been previously treated with AAV5 or the like.

[0031] As shown in this embodiment, the present invention may also enable AAV gene therapy, i.e., treatment of human patients exposed to AAV5, as is the case with other embodiments, where AAV5 antibody levels in an endemic, untreated human population enable efficient AAV5 gene therapy. For example, means and methods that can reduce the level of antibodies in the blood, and thereby reduce the level of anti-AAV5 antibodies, are known in the art. By employing such extracorporeal treatment of blood, such as removing antibodies from the blood, the titer of anti-AAV5 antibodies in the blood can be reduced to the same level observed in endemic exposure, i.e., in an endemic, untreated human population. Such methods are known in the art and may include, for example, plasmapheresis (Chicoine et al., Mol Ther 2014, Vol. 22, No. 2, 338-347). Therefore, by utilizing any method that can be employed to lower blood antibodies, including anti-AAV5 antibodies, human patients who were previously ineligible for treatment because they had been previously subjected to AAV5-based gene therapy can have their anti-AAV5 antibody titers reduced to a level that allows them to acquire anti-AAV5 antibody titers observed in endemic human populations and become eligible for AAV5-based gene therapy.

[0032] In another embodiment of the present invention, the AAV5 gene therapy vector described above comprises at least 10 11 It is administered in doses equivalent to individual capsids / kg body weight. It is understood that the observations made by the inventors regarding the presence of anti-AAV5 antibodies may be dose-dependent. In other words, the concentration and / or amount of anti-AAV5 antibodies at the dosage used does not impair transduction. For example, the amount of AAV5 gene therapy vector administered to a human patient in treatment to obtain a meaningful level of transgene expression far exceeds the amount of anti-AAV5 antibodies present in the blood. From this perspective, it may be considered that there is no upper limit. Nevertheless, the upper limit that can be considered is at most 10 16It is at a dosage corresponding to capsids / kg body weight. It is understood that the dosage may be set as the dosage per patient or the dosage per blood volume. At least 10 12 A dosage of capsids / kg body weight is based on an average body weight of about 85 kg and an average blood volume of 5 L, and is about 10 14 capsids per patient or about 10 13 capsids / L of the patient's blood volume. Therefore, no matter what dosage range is contemplated, these dosage ranges can be easily recalculated based on these parameters. The dosage is preferably at least 1×10 12 capsids / kg body weight, at least 5×10 12 capsids / kg body weight, or at least 1×10 13 capsids / kg body weight. The dosages used in the Examples section are about 5×10 13 capsids / kg body weight and about 2×10 14 capsids / kg body weight. The quantification of AAV capsid particle titer of AAV is easily determined and is well-known in the art (especially, Kohlbrenner et al., Hum Gene Ther Meth. June 2012, Vol. 23, No. 3: 198-203; Grimm et al., Gene Ther., Vol. 6, No. 7, pages 1322-1330, 1999).

[0033] The selected dosage may also be based on genome copies. Genome copy means the amount of vector genome contained in the AAV5 preparation. The gc titer of the AAV5 vector preparation can be easily determined by using qPCR to quantify the vector genome sequence. The AAV5 gene therapy vector is preferably used at a dosage corresponding to at least 5×10 11 gc / kg body weight. A dosage of at least 5×10 11 capsids / kg body weight is based on an average body weight of about 85 kg and an average blood volume of 5 L, and is about 5×10 12 gc per patient or about 10 12This is converted to gc / patient's blood volume in liters. Therefore, whatever dose range is intended, these dose ranges can be easily recalculated based on these parameters. The selected dosage is at least 1 × 10⁻⁶. 12 gc / kg body weight, at least 2 × 10 12 gc / kg body weight, or 4 × 10 12 It may also be gc / kg body weight. The dosage used in the Examples section is approximately 5 × 10 12 gc / kg body weight and approximately 2 × 10 13 This is gc / kg body weight. There is no upper limit, but the upper limit should be no more than 10 15 The dosage may be set to correspond to the amount of medication equivalent to gc / kg body weight.

[0034] As stated, the AAV5 gene therapy vector according to the present invention is intended for use in medical treatment. The transgene contained in the AAV virus vector according to the present invention is not limited to the present invention. Nevertheless, preferably and according to the examples, the therapeutic gene may include variants of human factor IX, such as those described in International Publication Nos. 2010029178, 1999003496, 2015086406, and 2010012451, which encode human factor IX as described in Nathwani et al., N Engl J Med 2011;365(25):2357~65 and Nathwani et al., BN Engl J Med 2014;371(21):1994~200 and are incorporated herein by reference in their entirety. In particular, the Examples section demonstrates that therapeutically significant amounts of protein can be obtained in human patients using the FIX-coded AAV5 vector. Such proteins may be useful, for example, in the treatment of hemophilia A or hemophilia B.

[0035] Therefore, the present invention provides an AAV5 gene therapy vector for use in human medical treatment, wherein the AAV5 gene therapy vector is used in the treatment of hemophilia B, and the amount of transgenic FIX protein acquired in plasma may be in the range of about 0.02 micrograms / ml to a maximum of about 5 ug / ml. Alternatively, when the AAV5 gene therapy vector is used in the treatment of hemophilia B patients with a severe phenotype, the patient may acquire a moderate or mild phenotype, or even a phenotype observed in healthy individuals, after treatment. Hemophilia B can be classified into three classes, each characterized by the presence of FIX at different plasma concentrations. In severe hemophilia B, plasma levels of FIX activity are below 1% of normal; in moderate morphology, levels are 1% to 5%; and in mild morphology, levels are 5% to 25% of normal levels. There are healthy carrier individuals with intermediate FIX activity levels of 25% to 50% of normal, but many carriers may have levels even higher than 50%.

[0036] Similarly, therapeutically effective amounts of other genes of interest can be expected to be well within reach of those skilled in the art. Therefore, the present invention is expected to be useful with respect to any transgene. Further suitable transgenes for delivery to the patient in viral vectors for gene therapy may be selected by those skilled in the art. These therapeutic nucleic acid sequences typically encode products (e.g., proteins or RNA) for in vivo or ex vivo administration and expression in the patient to treat hereditary or non-hereditary genetic defects, epigenetic disorders or diseases, or pathological conditions associated with dysregulation of gene products, for example, by replacing or correcting deletions. Such therapeutic genes desirable for the performance of gene therapy include, but are not limited to, the very low-density lipoprotein receptor gene (VLDL-R) for the treatment of familial hypercholesterolemia or familial combined hyperlipidemia, the cystic fibrosis transmembrane regulator gene (CFTR) for the treatment of cystic fibrosis, the DMD-Becker allele for the treatment of Duchenne muscular dystrophy, and several other genes that can be readily selected by those skilled in the art to treat specific disorders or diseases. In a preferred embodiment, the rAAV vector comprises a therapeutic protein or a transgene encoding an RNA such as miRNA. The therapeutic protein may be factor IX (preferably human factor IX), factor VIII (preferably human factor VIII), or lipoprotein lipase (LPL; e.g., LPL). S447XIt is preferable to select from the group consisting of the following (including variants such as; see International Publication No. 01 / 00220 A2), porphobilinogen deaminase (PBGD), very low-density lipoprotein receptor (VLDL-R), cystic fibrosis transmembrane conductance regulator (CFTR), Duchenne muscular dystrophy (DMD) Becker allele, hyperoxaluria (AGXT), N-acetyl-α-D-glucosaminidase (NaGlu), glial cell line-derived neurotrophic factor (GDNF), and S100A1 (also known as S100 calcium-binding protein A1, encoded in humans by the S100A1 gene). In a preferred embodiment, the therapeutic protein is factor IX, and more preferably human factor IX.

[0037] Alternatively, or in combination with any one of the embodiments described above, in a preferred embodiment, gene therapy is for treating, preventing, curing, and / or reversing a medical condition or disease, preferably a so-called rare disease, which is understood herein to be a rare disease affecting a small proportion of a population, for example, less than 1 in 1,500 people in the population, that is life-threatening, chronically debilitating, and / or inadequately treatable. Generally, rare diseases are genetic disorders and are therefore lifelong diseases, even if symptoms do not appear immediately. In preferred embodiments, such conditions or diseases include lipoprotein lipase deficiency (LPLD), hemophilia B, acute intermittent porphyria (AIP), Sanfilipo B syndrome, Parkinson's disease (PD), congestive heart failure (CHF), hemophilia A, Huntington's disease, Duchenne muscular dystrophy (DMD), Leber congenital amaurosis, X-linked severe combined immunodeficiency (SCID), adenosine deaminase-deficient severe combined immunodeficiency (ADA-SCID), adrenoleukodystrophy, chronic lymphocytic leukemia, acute lymphoblastic leukemia, multiple myeloma, cystic fibrosis, sickle cell disease, hyperlipoproteinemia type I, thalassemia, and Alzheimer's disease. The group is selected from the following: Heimer's disease, amyotrophic lateral sclerosis (ALS), epilepsy, Friedreich's ataxia, Fanconi anemia, Batten disease, wet AMD, alpha-antitrypsin-1, Pompe disease, SMA-1, drug-resistant non-small cell lung cancer, GM1 gangliosides, retinitis pigmentosa, homozygous familial hypercholesterolemia, lysosomal storage disorders, copper or iron storage disorders (e.g., Wilson's disease or Menkes disease), lysosomal acid lipase deficiency, hyperoxaluria, Gaucher disease, Hurler's disease, adenosine deaminase deficiency, glycogen storage disorders, and retinal degenerative diseases (e.g., RPE65 deficiency, colloideremia).

[0038] In further embodiments, the AAV5 gene therapy vector is intended for use in human medical treatment according to the present invention, and such use includes administration into the bloodstream, for example, administration of the AAV5 gene therapy vector into the bloodstream. The blood may contain anti-AAV5 antibodies, and in particular, delivery routes via the bloodstream, for example, intravascular injection or injection, are intended. Delivery via the bloodstream allows for delivery of the AAV5 vector to target tissue. Such delivery to target tissue may occur by systemic delivery. The present invention is not limited to delivery into the bloodstream. In fact, conventional and pharmaceutically acceptable routes of administration that can be intended include direct delivery to target organs, tissues, or sites (e.g., liver or CNS), intranasal, intravenous, intramuscular, subcutaneous, intradermal, oral, and other parental delivery routes. However, a preferred target tissue that can be intended is the liver. Therefore, it is most preferred that the AAV5 gene therapy vector delivers the vector's transgene to the liver via a delivery route via the bloodstream. As described, administering a sufficient amount of the AAV5 viral vector to transfect the desired cells and provide sufficient levels of transduction and expression of the selected transgene provides therapeutic benefits without excessive adverse effects that can be judged by those skilled in the art of medicine, or with medically acceptable physiological effects. The route of administration may also be combined as needed. The dosage of the rAAV vector (i.e., the AAV5 gene therapy vector) is thought to depend primarily on factors such as the disease being treated, the selected gene, the patient's age, weight, and health status, and therefore may vary considerably among patients.

[0039] The AAV5 gene therapy vector for use in human medical treatment according to the present invention is preferably an AAV5 gene therapy vector produced in insect cells. Without being constrained by theory, AAV capsids produced in insect cells may differ from those produced in mammalian cells, and the method of production may play a role in the immune profile associated with the AAV vector. This difference may relate to glycosylation or other post-translational modifications. Furthermore, mammalian cell-based production may have the disadvantage that rep and cap expression constructs are contained in the AAV capsid administered to the patient, resulting in the transfer of even small amounts of rep and cap expression constructs into the human subject. Expression of AAV rep and cap in human patients may be detrimental from an immunological standpoint, particularly in human patients who will test positive for anti-AAV5 antibodies. Therefore, it may be preferable that the AAV5 viral vector to be administered to human patients is produced in insect cells. Manufacturing based on insect cells is well established and includes, but is not limited to, the means and methods described in International Publication Nos. 2007046703, 2007148971, 2009014445, 2009104964, 03042361, 2008024998, and 2010114948, which are incorporated herein by reference.

[0040] In another embodiment, A method for determining whether a human patient is eligible to receive medical treatment using an AAV5 gene therapy vector, Steps to prepare serum samples derived from human patients, Steps to determine the anti-AAV5 antibody titer, If the total anti-AAV5 antibody titer is within the range of 0.02 to 5 as determined using the total anti-AAV5 antibody (TAb) assay described in the examples, the patient may be considered eligible for medical treatment. A method including this is provided.

[0041] Optionally, this method follows Steps to administer the AAV5 gene therapy vector to eligible human patients. Includes.

[0042] Any of the boundaries and limitations described above with respect to embodiments relating to the medical use of AAV5 gene therapy factors are understood to also apply to any of the methods described herein, for example, for methods of delivering AAV5 gene therapy vectors or for determining eligibility. The total anti-AAV5 antibody titer is preferably in the range of 0.02 to 4, 0.02 to 3, or 0.02 to 2, as determined using the total anti-AAV5 antibody (TAb) described in the examples.

[0043] In another embodiment, a method for determining whether a human patient is eligible to receive medical treatment using an AAV5 gene therapy vector is: Steps to prepare serum samples derived from human patients, Steps to determine the anti-AAV5 antibody titer, If the titer of the neutralizing anti-AAV5 antibody is within the range of 3 to 10,000 as determined using the neutralizing anti-AAV5 antibody (NAb) assay described in the examples, the patient may be considered eligible for medical treatment. Includes.

[0044] Optionally, this method follows Steps to administer the AAV5 gene therapy vector to eligible human patients. Includes.

[0045] The anti-AAV5 antibody titer is preferably within the range of 3 to 5,000, 3 to 3,000, or 3 to 1,000, as determined using the neutralizing anti-AAV5 antibody (NAb) described in the examples.

[0046] It should be understood that the eligibility criteria described above are not the only criteria that may be used to select AAV5 gene therapy. Therefore, if a human patient meets all other criteria, the eligibility of the human patient will be determined by the anti-AAV5 antibody scale. In another embodiment, a method for treating a human being comprises the step of administering an effective amount of an AAV5 gene therapy vector to a human being in need thereof, The aforementioned human was not subjected to pre-screening using an assay to determine anti-AAV5 antibodies. A method is provided in which the human being has not been subjected to medical treatment using an AAV5 gene therapy vector prior to the medical treatment.

[0047] In a further embodiment, a method for treating a human being comprises the step of administering an effective amount of AAV5 gene therapy vector to a human being in need thereof, The aforementioned human subjects were subjected to pre-screening using an assay to determine anti-AAV5 antibodies. The aforementioned human being had not been subjected to medical treatment using the AAV5 gene therapy vector prior to the aforementioned medical treatment. The method is provided wherein the human has an anti-AAV5 antibody level that corresponds at most to the 95th percentile of the anti-AAV5 antibody levels observed in the human population.

[0048] In another further embodiment, a method for delivering a gene to a human, comprising the step of administering an effective amount of an AAV5 gene therapy vector to a human in need thereof, The aforementioned human subjects were subjected to pre-screening using an assay to determine anti-AAV5 antibodies. The aforementioned human being had not been subjected to medical treatment using the AAV5 gene therapy vector prior to the aforementioned medical treatment. The method is provided wherein the human has an anti-AAV5 antibody level that corresponds at most to the 95th percentile of the anti-AAV5 antibody levels observed in the human population.

[0049] In another embodiment, a method for delivering a gene to a human, comprising the step of administering an effective amount of an AAV5 gene therapy vector to a human in need thereof, The aforementioned human was not subjected to pre-screening using an assay to determine anti-AAV5 antibodies. A method is provided in which the human being has not been subjected to medical treatment using an AAV5 gene therapy vector prior to the medical treatment.

[0050] Alternatively, or in combination with any one of the embodiments described above, in a preferred embodiment, the AAV5 vector composition further comprises a pharmaceutically acceptable carrier, diluent, solubilizer, filler, preservative, and / or excipient. Preferably, the rAAV vector carrying the therapeutic gene can be administered to a patient suspended in a biocompatible solution or a pharmaceutically acceptable delivery vehicle. Suitable vehicles include sterile saline. Other aqueous and non-aqueous isotonic sterile injection solutions and aqueous and non-aqueous sterile suspensions, which are known to be pharmaceutically acceptable carriers and are well known to those skilled in the art, may be employed for this purpose. The viral vector is administered to a human patient in sufficient quantities as described above to transfect the desired cells and provide sufficient levels of transduction and expression of the selected transgene, thereby providing therapeutic benefits without excessive adverse effects as determined by those skilled in the art of medicine, or with pharmaceutically acceptable physiological effects. [Examples]

[0051] Survey design and participants A multinational, open-label, dose-escalation Phase 1 / 2 study was conducted, including adult males with severe (FIX < 1 IU / dL) or moderate to severe (FIX ≤ 2 IU / dL) hemophilia B who required either 1) continuous fix prophylaxis or 2) fix on demand and experienced ≥ 4 bleeding episodes or hemophilic arthropathy per year. Further details of the study can be found on the NIH clinicaltrials.gov website (NCT02396342). The study was authorized by institutional review boards / ethics boards at each center. All participants provided written informed consent. The study was conducted in accordance with the principles of the Declaration of Helsinki and Good Clinical Practice.

[0052] In this study, we used AAV5 vectors incorporating codon-optimized wild-type hFIX genes under the control of the liver-specific promoter LP1 (Nathwani et al., N Engl J Med 2011;365(25):2357~65, and Nathwani et al., BN Engl J Med 2014;371(21):1994~2004). The vectors were manufactured using a baculovirus expression system in accordance with Good Manufacturing Practices for pharmaceuticals and quasi-drugs. The vector genome copy titer (gc) was determined using qPCR. The capsid:gc ratio was approximately 10, meaning the amount of capsid was approximately 10 times the amount of genome copies. Capsid titer can be determined by high-performance liquid-size exclusion chromatography (HPL-SEC) with UV absorption detection. The method is based on the SEC column, which is selected for its ability to separate AAV particles from smaller matrix components. In this method, a calibration curve is created using AAV vector preparations with known total particle concentrations. The calibration curve plots the total amount of injected particles against the response data. Using the recovered AAV peak area and the calibration curve, the amount of sample particles injected is calculated by interpolation. The AAV5 vector was administered as a single 30-minute peripheral intravenous infusion. Participants were treated in two consecutive escalating dose cohorts: Cohort 1 (n=5, participants 1-5) was 5 × 10⁶ 12 After receiving gc / kg, Cohort 2 (n=5, participants 6-10) was 2 × 10 13 gc / kg was administered. Cohort 1 consisted of adult males with a mean age of 69 years (35–72) and a mean weight of 84.5 kg (71.2–89.1), and Cohort 2 consisted of adult males with a mean age of 35 years (33–46) and a mean weight of 84.0 kg (71.4–96.0). Efficacy outcome measurements included FIX plasma activity measurement. In addition, serum from the subjects was obtained for neutralizing AAV5 antibody titer (NAb titer) and total AAV5 antibody titer (TAb) analysis.

[0053] Control serum from healthy donors was commercially obtained from SeraLab (West Sussex, UK). All information provided regarding these serums is listed below.

[0054] [Table 1]

[0055] Neutralizing AAV5 antibody (NAb) titer NAb levels in human serum were assessed using a highly sensitive in vitro assay with AAV5 (AAV5-luc) cells containing the transgene luciferase and the human embryonic kidney cell line HEK293T (ATCC 11.268). Transgene expression was revealed by the addition of a luciferin analog.

[0056] Materials used: HEK293T cells (HEK293T / ATCC 11.268) DMEM containing phenol red (Gibco, reference #31966) / 10% FBS (Greiner, reference #758093) / 1% PenStrep (Gibco, reference #15140) DMEM without phenol red (Gibco, reference #21063) / 1% Pen-Strep (Gibco, reference #15140) 1×PBS- / -(Gibco, reference #14190) 1x Trypsin EDTA (Gibco, see #25200) Poly-L-lysine (PLL) solution (2.5%) (Sigma-Aldrich, reference #8920-100) 96-well flat-bottom black culture plate (costar, reference #3916) Transparent 96-well flat-bottom plate (corning, see #3596) ONE-Glo Luciferase Assay System (Promega, see #E6120) Glo Lysis Buffer, 1x (Promega, see #E2661) AAV5-CMV-luc (for example, using AAV5-CMV-73QlucHtt from PKO, titer: 4e13gc / ml)

[0057] Basically, 0.5 × 10 5 HEK293T cells were seeded into black and clear 96-well plates by adding 100 μl / well of HEK293T cells to DMEM (containing phenol red / 10% FBS and 1% P / S (penicillin / streptomycin)) at a concentration of individual cells / well. The cells were incubated overnight.

[0058] The following day, serial dilutions of plasma were prepared in a clear 96-well plate in culture medium (DMEM / 1% PS, phenol red-free / 10% FBS). After the addition of the virus (see below), the final plasma dilutions obtained were 2, 4, 8, 16, 32, 64, 128, 256, 512, and 1024.

[0059] Dilutions are prepared by adding 140 μl of medium to the wells designated A2 to A11 (negative control wells), and 70 μl of medium is added to the remaining rows in columns 3, 4, 5, 6, 7, 8, 9, 10, and 11 of the plate, as well as to rows H2 to H11 (positive control).

[0060] Plasma samples were added to wells B2, C2, D2, E2, F2, and G2 (140 μl / well), resulting in the first dilution: 1. Serial dilutions of plasma were then performed across the plate by moving 70 μl each from column 2 to column 3 (dilution 2), 3 to 4 (dilution 4), 4 to 5 (dilution 8), 5 to 6 (dilution 16), 6 to 7 (dilution 32), 7 to 8 (dilution 64), 8 to 9 (dilution 128), 9 to 10 (dilution 256), and 10 to 11 (dilution 512), after which 70 μl from column 11 was discarded. AAV5-CMV-73QlucHtt was then incubated in medium (DMEM / 1% PS, phenol red-free / 10% FBS) for 6 × 10⁶ 9 Prepare with gc / ml. Next, prepare 70 μl / well in 6 × 10 9Add the gc / ml AAV5(160)-CMV-73QlucHtt virus dilution to a plasma dilution plate, excluding wells A2-A11 (negative control). Carefully place the plate in a plate shaker at 300 rpm for 2 minutes. Then, incubate the plate at 4°C for 1 hour.

[0061] The culture medium was removed from the black 96-well plate (containing HEK293T cells) prepared the previous day, and replaced with 100 μl / well of prepared plasma dilution in the black plate containing Hek293T cells by pipetting from the clear plate. These plates were incubated at 37°C for 16–20 hours. The following day, the cells were equilibrated at room temperature, and the medium was removed. The cells were rinsed once with 1×PBS- / - (100 μl / well), then 100 μl / well of Glo Lysis Buffer was added to the plate, and incubated at room temperature for 5 minutes to induce lysis. Subsequently, 100 μl / well of reagent from the ONE-Glo Luciferase Assay System was added (reagent prepared according to the manufacturer's instructions). After at least 3 minutes, the plate was measured using the ONE-Glo protocol on a GloMax Discover instrument. After subtracting background activity, the percentage of neutralization for each serum dilution is calculated, and then the anti-AAV5 neutralizing antibody titer is determined using LabKey software analysis to fit the curve to the neutralization profile. LabKey then uses this curve to calculate the neutralizing antibody titer against the selected benchmark, area under the curve (AUC), and error estimate. Curve fitting was calculated using a four-parameter method. LabKey calculates the IC50, which is the dilution in which the antibody inhibits transduction by 50%. LabKey also calculates a "point-based" titer according to Johnson and Byington, Techniques in HIV Research. New York, NY: Stockton Press, 1990:71-76. This calculation is performed by linearly interpolating between two replicates on both sides of the target neutralization percentage. Each run included a positive control (a well containing AAV5-LUC but without sample serum), a negative control (a well containing only culture medium, without sample serum or AAV5-LUC), and negative control sample serum (thermal-inactivated FBS) to assess the specificity of AAV5-LUC neutralization. FBS should not possess anti-AAV5 neutralizing properties when measured as a sample.

[0062] Anti-AAV5 antibody titer Quantification of all human antibodies against AAV5 was based on an ELISA assay using plates coated with a specific capsid. The presence of all human antibodies specific to the AAV5 capsid was revealed using protein A peroxidase. ELISA plates (Nunc MaxiSorp plates, reference: 456537, Thermo Scientific) were coated overnight at 4°C with 100 ng / well antigen (AAV5 cap) in carbonate buffer. The following day, the plates were washed three times with PBS tween-20 (PBSt) to remove any remaining antigen and blocked with blocking solution (PBS + 3% FBS) to prevent nonspecific binding. After washing three times with 200 μL of PBSt, human serum dilutions in PBSt were added in a final volume of 100 μL, starting at 1:9 and followed by a dilution series of 1:3. All samples were tested in pairs. A negative control without human serum was included in each plate. Serum dilutions were incubated at 37°C for 2 hours. Afterward, the serum was removed, the plate was washed three times with PBSt, and 100 μL of protein A peroxidase diluted 1:10,000 was added to the blocking solution for 1 hour. The plate was washed three times with PBSt, the reaction was clarified with TMB substrate, and stopped with H2SO42N after 30 minutes. Absorbance was read at 450 nm using a microplate reader. The total antibody titer was calculated as a serum dilution, and the total antibody titer had an absorbance 5 times higher than that of the negative control.

[0063] Results and Discussion All human patients in both cohorts presented a meaningful improvement in FIX activity, with most showing improvement through a phenotypic shift from severe to mild (Table 2), resulting in a substantial reduction or even absence of prophylactic FIX protein use. Variation was observed between patients and between cohorts in FIX activity levels. This variation in FIX activity levels did not correlate with the NAb or TAb status of human patients.

[0064] The previously reported prevalence rate of TAbs against AAV5 (40%, Boutin et al., Hum Gene Ther. June 2010, 21(6):704-712) was in general agreement with the results of this analysis (30%). Results obtained using luciferase-based NAb assays (see Figures 1 and 2) showed that 14 out of 50 screened control serums (28%) showed restored positive signals, suggesting a similar prevalence rate of AAV5 (neutralizing) antibodies, which is consistent with recent studies (Li C et al., Gene Ther. March 2012; 19(3):288-94). Results from serums obtained from human patients prior to gene therapy treatment were similarly consistent with recent studies, with 3 out of 10 serums found to be positive in both NAb and Tab assays (30%). The total antibodies assessed by ELISA and the neutralizing antibodies assessed by luciferase-based assays are closely correlated, suggesting that both assays detect the same entities (see Figure 2A).

[0065] Furthermore, the presence of neutralizing antibody titers after treatment was also tested, and approximately 10 6 It was found that antibody titers were within or above that range. Therefore, antibody titers found in endemic, untreated humans were considerably outside the range of titers observed in human patients subjected to AAV5-based gene therapy. Furthermore, patients with pre-existing AAV5 NAbs showed a rapid increase in IgG characteristic of an immune boost upon administration of AAV-FIX, in contrast to patients without NAbs, who showed a rapid and transient increase in IgM followed by an increase in IgG, typical of initial exposure to the antigen. In addition, there was no evidence of elevated ALT (alanine aminotransferase) or capsid-specific T cell activation in treated patients with pre-existing NAbs. Therefore, administration of AAV5-based gene therapy in endemic, pre-existing NAb-present patients was well-tolerated without elevated ALT or T cell activation.

[0066] In conclusion, the presence of anti-AAV5 antibodies detected in vitro by either the NAb assay or the TAB assay did not predict or indicate impairment of in vivo transduction. There was no clear correlation between the presence of Nab before therapy and the post-therapy FIX level resulting from AAV5 FIX gene transfer. Notably, the highest responder in Cohort 1, which received lower doses of AAV5 vector, also had the highest levels of detected NAb and TAb antibodies. The range of anti-AAV5 titers observed in the healthy population indicates that antibody levels in healthy populations not subjected to AAV5 gene therapy do not impair in vivo AAV5 transduction. This is because the highest titer observed in the healthy population was within the range of the highest titer observed in patient 5 of Cohort 1. Therefore, it is considered feasible not to test for the presence of anti-AAV5 antibodies in untreated populations before treatment with AAV5 gene therapy vectors.

[0067] [Table 2]

[0068] Further embodiments are as follows: [Embodiment 1] An AAV5 gene therapy vector for use in the medical treatment of humans, wherein the human is not subjected to pre-screening using an assay to determine anti-AAV5 antibodies, and the human is not subjected to medical treatment using the AAV5 gene therapy vector prior to the medical treatment. [Embodiment 2] An AAV5 gene therapy vector for use in the medical treatment of humans, wherein the human is subjected to pre-screening using an assay to determine anti-AAV5 antibodies, the human has not been subjected to medical treatment using the AAV5 gene therapy vector prior to the medical treatment, and the human has an anti-AAV5 antibody level that corresponds at most to the 95th percentile of anti-AAV5 antibody levels observed in human populations. [Embodiment 3] The AAV5 gene therapy vector for use in the medical treatment of a human, as described in Embodiment 2, wherein the human has shown a positive test result against the anti-AAV5 antibody (body). [Embodiment 4] at least 10 12 An AAV5 gene therapy vector for use in human medical treatment according to any one of Embodiments 1 to 3, administered in a dosage equivalent to individual capsids / kg. [Embodiment 5] at least 10 12 An AAV5 gene therapy vector for use in human medical treatment according to any one of Embodiments 1 to 4, used in a dosage equivalent to gc / kg body weight. [Embodiment 6] An AAV5 gene therapy vector for use in human medical treatment according to any one of Embodiments 1 to 5, used in the treatment of a disease selected from the group consisting of hemophilia A or hemophilia B. [Embodiment 7] An AAV5 gene therapy vector for use in human medical treatment according to any one of Embodiments 1 to 6, which is used in the treatment of hemophilia and encodes the FIX protein or a variant thereof. [Embodiment 8] The aforementioned use includes administration into the bloodstream, and is an AAV5 gene therapy vector for use in human medical treatment according to any one of Embodiments 1 to 7. [Embodiment 9] The aforementioned use comprises delivery of the vector to the liver, and is an AAV gene therapy vector for use in human medical treatment according to any one of Embodiments 1 to 8. [Embodiment 10] An AAV5 gene therapy vector for use in human medical treatment according to any one of Embodiments 1 to 9, produced in insect cells. [Embodiment 11] A method for determining whether a human patient is eligible to receive medical treatment using an AAV5 gene therapy vector, Steps to prepare serum samples derived from human patients, Steps to determine the anti-AAV5 antibody titer, If the total anti-AAV5 antibody titer is within the range of 0.02 to 5 as determined using the total anti-AAV5 (TAb) assay described in the examples, the patient may be considered eligible for medical treatment. A method that includes this. [Embodiment 12] A method for determining whether a human patient is eligible to receive medical treatment using an AAV5 gene therapy vector, Steps to prepare serum samples derived from human patients, Steps to determine the anti-AAV5 antibody titer, If the anti-AAV5 antibody titer is within the range of 3 to 5,000 as determined by the neutralizing anti-AAV5 antibody assay, which is determined by the NAb assay described in the examples, the patient may be considered eligible for medical treatment. A method that includes this.

Claims

1. A pharmaceutical composition comprising an AAV5 gene therapy vector for use in human medical treatment, The aforementioned human was not subjected to pre-screening using an assay to determine anti-AAV5 antibodies. The aforementioned human being has not been subjected to medical treatment using an AAV5 gene therapy vector prior to the aforementioned medical treatment, and the pharmaceutical composition.

2. at least 10 １２ The pharmaceutical composition according to claim 1, administered in a dosage equivalent to individual capsids / kg.

3. at least 10 １２ The pharmaceutical composition according to claim 1 or 2, used in a dosage equivalent to gc / kg body weight.

4. at least 10 １１ The pharmaceutical composition according to claim 1, administered in a dosage equivalent to individual capsids / kg.

5. At least 5 x 10 １１ The pharmaceutical composition according to claim 1 or 4, used in a dosage equivalent to gc / kg body weight.

6. A pharmaceutical composition according to any one of claims 1 to 5, used in the treatment of a disease selected from the group consisting of hemophilia A or hemophilia B.

7. A pharmaceutical composition according to any one of claims 1 to 6, used in the treatment of hemophilia, wherein the AAV5 gene therapy vector encodes the FIX protein or a variant thereof.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the use includes administration into the bloodstream.

9. The pharmaceutical composition according to any one of claims 1 to 8, wherein the use comprises delivery of the AAV5 gene therapy vector to the liver.

10. A pharmaceutical composition according to any one of claims 1 to 5, used in the treatment of Huntington's disease.

11. The pharmaceutical composition according to claim 10, wherein the AAV5 gene therapy vector encodes a miRNA.

12. The pharmaceutical composition according to claim 10 or 11, wherein the use includes administration to the central nervous system (CNS).

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein the AAV5 gene therapy vector is produced in insect cells.

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