Means and methods for AAV gene therapy in humans

Abstract

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 that are intended for use in therapy.

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 that are intended for use in therapy. [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 dividing and non-dividing human cells. The wild-type AAV viral genome integrates into a single chromosomal site in the host cell genome, and most importantly, even though AAV is present in many humans, AAV has not been 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. The clinical trials and approval of numerous gene therapy drugs in Europe, such as Alipogene tiparvovec (Glybera®, uniQure), hold the promise of AAV becoming mainstream in clinical practice.

[0003] One major challenge to 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 viral capsid proteins can reduce the efficiency of gene transfer using AAV of the same serotype.

[0004] Relatively low endemic NAB titers have been observed for the AAV5 serotype in humans compared with other serotypes (Boutin et al., Hum Gene Ther 2010, 21:704-712). It has been previously reported that such low endemic NAB titers affect transduction and result in severely reduced transgene expression in human treatment with AAV (Manno et al., Nature Medicine 2006, 12(3), 342-347). Therefore, the general consensus in the field is to completely avoid treating patients with NAB titers. Therefore, current clinical practice for pre-existing immunity involves screening human patients for exclusion if they have 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 attempted to reduce NAb formation immediately after the first dose to allow for 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 the use of plasma exchange and immunosuppressive regimens have been suggested to overcome pre-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 with limited success.

[0005] Thus, there is a need in the art to enable the administration of rAAV gene therapy vectors in human patients who have or may be suspected of having AAV neutralizing antibodies. Summary of the Invention [Problem to be solved by the invention]

[0006] The present inventors have now surprisingly found that, in contrast to what the state of the art suggests, particularly with regard to AAV5 gene therapy vectors, human patients with endemic pre-existing anti-AAV5 antibodies (i.e., pre-existing anti-AAV5 antibodies resulting from endemic exposure) may be considered eligible for treatment. This finding contrasts with the prior art belief that the presence of neutralizing antibodies should be considered an exclusion criterion for patients participating in, for example, clinical trials. In other words, the state of the art belief is that patients with antibodies to AAV5, and more particularly endemic pre-existing antibodies to AAV5, are considered ineligible for treatment with AAV5 gene therapy vectors. The surprising finding disclosed herein that human patients with endemic pre-existing anti-AAV5 antibodies may be considered eligible for treatment is found to pertain not only to a subpopulation of human patients who are found to have, for example, very low levels of pre-existing anti-AAV5 antibodies, but rather to pertain essentially to most, if not all, patients in the human population who score positive for pre-existing anti-AAV5 antibodies and who have never before been subjected to any AAV5 gene therapy.

[0007] Thus, in one aspect, 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 with an assay to determine anti-AAV5 antibodies, and wherein the human has not been subjected to medical treatment with an AAV5 gene therapy vector prior to the medical treatment. In other words, an AAV5 gene therapy vector for use in the medical treatment of a human, wherein the anti-AAV5 antibody status is unknown, and wherein the human has not been subjected to medical treatment with an AAV5 gene therapy vector prior to the medical treatment, is provided. According to one aspect, the present invention may enable treating patients not previously treated with an AAV5 gene therapy vector or including patients in clinical trials without prior medical treatment, screening for the presence of anti-AAV5 antibodies.

[0008] In a further aspect, an AAV5 gene therapy vector is provided for use in medical treatment of a human, wherein the human has been 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 to at most the 100th percentile, preferably at most the 95th percentile, of anti-AAV5 antibody levels observed in the human population. Preferably, the human patient tested is positive for anti-AAV5 antibodies. [Brief explanation of the drawings]

[0009] [Figure 1A] NAb assay results for pre-treatment samples. A: Graph of neutralization results for 10 pre-dose samples. The 50 percent mark is shown as a dotted line. [Figure 1B]B: Curve fitting results for three positive samples 3, 4, and 5. A four-parameter curve was fitted by nonlinear regression. The titer was calculated as the theoretical dilution at which the fitted curve passed the 50% mark (shown above the horizontal axis). [Figure 2A] Graph of AAV5 neutralizing antibodies versus total anti-AAV5 antibodies. Neutralizing (NAb) titers versus total (TAb) ELISA results reported in a population screening study. Each open symbol represents paired NAb and TAb results for one healthy individual. NAb and TAb results for treated patients are shown overlaid (▲ (black triangle), with study subjects 3, 4, and 5 designated). [Figure 2B] Graph of AAV5 NAb titer versus FIX levels. Percentage of FIX activity in Cohort 1 after dosing is plotted against pre-dose NAb titer. [Figure 3]

[0023] Figure 1. Diagram of the AAV NAb titer scale. Percentiles of the human population (50 subjects) are depicted with respect to NAb titers to AAV5. Note that the NAb titers observed in the human population are quite 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 a hybrid VP1 sequence is depicted, consisting of an N-terminal AAV2-derived VP1 sequence (underlined) linked to an AAV5-derived VP2 and VP3 coding sequence. Thus, the VP1 protein is a hybrid AAV2 / AAV5 capsid protein. Expression constructs used for AAV capsids encoding hybrid VP1 may also encode VP2 and VP3 sequences that are wild-type sequence AAV5 VP2 and VP3 proteins rather than 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 the modification.

[0010] definition "AAV vector" refers to a recombinant adeno-associated virus (AAV) vector derived from wild-type AAV by using molecular methods. AAV vectors are distinguished from wild-type (wt) AAV vectors because 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 containing a combination of AAV capsid and AAV genomic ITRs can be produced using methods known in the art, such as those described in 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 the production of rAAV was described by Urabe et al. (Urabe et al.

[2002] Human Gene Therapy 13(16):1935-1943), which consisted of three baculoviruses, named Bac-Rep, Bac-cap, and Bac-vec, whose co-infection into insect cells, such as SF9, resulted in the production of rAAV. The properties of the produced rAAV, i.e., physical and molecular characteristics, including potency, were not significantly different from those of rAAV produced in mammalian cells (Urabe

[2002] , supra). The original baculovirus system by Urabe (2002, supra) has been further developed (see, e.g., Kohlbrenner et al. (2005) Molecular Therapy 12(6):1217-1225; Urabe et al. (2006) Journal of Virology 80(4):1874-1885; WO 2007 / 046703; WO 2007 / 148971; WO 2009 / 014445, and WO 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. A transgene includes 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 an expression control element such as a promoter to drive transcription). The transgene is preferably inserted between inverted terminal repeat (ITR) sequences. The transgene may also be an expression construct comprising an expression control element, such as a promoter or transcriptional 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 a target cell with an rAAV vector of the invention results in the transfer of the transgene contained in the vector into the transduced cell. "Host cell" or "target cell" refers to the cell into which DNA delivery occurs, e.g., a synovial cell or synovial cell of an individual. AAV vectors can transduce both dividing and non-dividing cells.

[0014] A "gene" or "coding sequence" refers to a DNA or RNA region that "encodes" a particular protein. A coding sequence, when placed under the control of an appropriate regulatory region, such as a promoter, is transcribed (DNA) and translated (RNA) into a polypeptide. A gene may contain several operably linked fragments, such as a promoter, 5' leader sequence, introns, coding sequence, and 3' untranslated sequence, including a polyadenylation site or signal sequence. A chimeric or recombinant gene is a gene that is not normally found in nature, for example, a gene that is not naturally associated with a promoter and some or all of the transcribed DNA region. "Expression of a gene" 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 aligning two peptide or two nucleotide sequences using a global or local alignment algorithm, depending on the length of the two sequences. Sequences of similar length are preferably aligned using a global alignment algorithm (e.g., Needleman Wunsch) that optimally aligns the sequences throughout their entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g., Smith Waterman). Sequences may then be referred to as "substantially identical" or "essentially similar" if they share at least a certain minimum percentage of sequence identity (defined below) (e.g., when optimally aligned using the GAP or BESTFIT programs with default parameters). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over the entire length (full length) of the two sequences, maximizing the number of matches and minimizing the number of gaps. When two sequences have similar lengths, global alignment is appropriately used to determine sequence identity. Generally, the GAP default parameters are used, with a gap creation penalty of 50 (nucleotides) / 8 (proteins) and a gap extension penalty of 3 (nucleotides) / 2 (proteins). For nucleotides, the default scoring matrix used is nwsgapdna, and for proteins, the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919).Sequence alignment and scoring for percentage sequence identity 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 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 for GAP above or default settings (for both "needle" and "water", and for both protein and DNA alignments, the default gap opening penalty is 10.0, the default gap extension penalty is 0.5; the default scoring matrix is ​​Blosum62 for proteins and DNAFull for DNA). When sequences have substantially different overall lengths, local alignments, such as those using the Smith Waterman algorithm, are preferred. Alternatively, percentage similarity or identity can be determined by searching against public databases using algorithms such as FASTA, BLAST, etc.

[0016] As used herein, "gene therapy" refers to the insertion of a nucleic acid sequence (e.g., a transgene, as defined herein) into an individual's cells and / or tissues to treat a disease. A transgene can be a functional mutant allele that replaces or complements a defective allele. Gene therapy also includes the insertion of a transgene that inhibits, reduces, or decreases the expression, activity, or function of an endogenous gene or protein, such as a naturally inhibitory, i.e., undesirable, or abnormal (e.g., pathogenic), gene or protein. 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 amenable to gene therapy. Thus, an AAV5 gene therapy vector refers to an AAV5 vector for use in gene therapy.

[0017] In this document and in the claims hereto, the verb "comprise" and its conjugations are used in the open-ended sense of said word, meaning that items following said word are included, but items not specifically listed are not excluded.

[0018] Additionally, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires that there is one and only one element. Thus, the indefinite article "a" or "an" normally means "at least one."

[0019] The word "approximately" or "about," when used in connection with a numerical value (approximately 10, about 10), preferably means that the value may range from a given value of 10 to 10% greater or less than that value. DETAILED DESCRIPTION OF THE INVENTION

[0020] As noted, it has surprisingly been found that, particularly with regard to AAV5 gene therapy vectors, human patients with endemic pre-existing anti-AAV5 antibodies can be considered eligible for treatment. This finding contrasts with the commonly held belief that the presence of pre-existing anti-AAV antibodies to a particular serotype would preclude gene therapy treatment using that serotype. Without being bound by theory, AAV5 may be a serotype with relatively low endemic pre-existing anti-AAV5 antibody titers 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 route of infection, which may or may not involve helper virus co-infection. Furthermore, AAV5 is most divergent from and phylogenetically separate from other primate AAV serotypes, which may also contribute to the relatively low titers. Regardless of the underlying origin of the present invention, this divergence from other AAV serotypes allows most, if not all, members of the human population to be eligible for treatment using gene therapy or the like based on the AAV5 serotype. These individuals include the subset of the human population that is negative for anti-AAV5 antibodies, as well as the subset of the human population that is found to be positive for anti-AAV5 antibodies, but do not include the (currently) very small subset of the human population that is being treated with AAV5 gene therapy or the like, in which high titers of anti-AAV5 antibodies are observed (approximately 10% higher than those in endemic AAV5-infected individuals) such that these individuals would not be eligible for treatment with AAV5 gene therapy vectors or the like. 4 or higher).

[0021] Thus, in a first aspect of the present invention, there is provided an AAV5 gene therapy vector for use in medical treatment of a human, wherein the human 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.

[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 in GenBank (Accession No. 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 wild-type AAV5 amino acid sequences. 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 for which sequence identity can be determined is shown in FIG. 4. Such sequences may be naturally occurring sequences of AAV viruses that fall outside the phylogenetic perspective of the AAV5 clade (eg, as depicted in Figure 4).

[0023] A "serotype" is traditionally defined based on the lack of cross-reactivity between antibodies to one virus compared to another. Such differences in cross-reactivity are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to 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 all existing serotype-specific sera characterized for neutralizing activity, and no antibodies have been found that neutralize the virus of interest. As more naturally occurring virus isolates are discovered and capsid variants are created, they may or may not be serologically distinct from any of the currently existing serotypes. For convenience, the AAV5 serotype includes AAVs with capsid sequence modifications not characterized as distinct serotypes, which may also constitute subgroups or variants of the AAV5 serotype. Such variants generally share substantial sequence identity.

[0024] Non-natural capsid sequences are also contemplated in accordance with the present invention. For example, the serum-exposed (exposed to the outside world) amino acid sequence may be derived from one serotype, while the non-exposed amino acid sequence within the capsid may be derived from another serotype and / or may be more open to variation. As the crystal structure of AAV5 is known (e.g., Govindasamy et al., J. Virol. October 2013, Vol. 87, No. 20: 11187-11199), non-exposed sequences, e.g., internal to the AAV5 capsid, may be replaced with sequences from other serotypes and / or may be more open to variation. For example, the VP1 amino acid sequence, which is not contained in VP2 and VP3, may be placed internally. This sequence may be derived from, for example, serotype 2, while the VP2 and VP3 amino acid sequences may be entirely based on AAV5 (see, e.g., Figure 5). Such AAV5 gene therapy vector capsids have serotype and neutralizing antibody profiles indistinguishable from intact wild-type capsids (see, inter alia, WO 2000028004 and Urabe et al., J Virol, February 2006, Vol. 80, No. 4, pp. 1874-1885). Such non-native capsid sequences are also understood to be hybrid sequences, and such hybrid vectors are AAV5 gene therapy vectors according to the present invention. Furthermore, AAV5 capsid sequences can also have one or more amino acids inserted or replaced to enhance vector production and / or efficacy, such as those described in, inter alia, WO 2015137802 and shown, for example, in FIG. 6. Such slightly modified AAV5 capsids may also be considered to be of the AAV5 serotype.

[0025] An AAV5 vector or AAV5 gene therapy vector according to the present invention is understood to refer to an AAV5 vector capsid containing a vector genome with a gene of interest contained between AAV inverted repeats, which may, but need not, be AAV5 ITRs. Thus, an AAV5 vector according to the present invention is a delivery vehicle capable of delivering its payload, a vector genome with a transgene, e.g., a transgene that may be beneficial to humans, to its target cells, e.g., liver cells or cardiomyocytes. Thus, AAV5 vectors may be useful in the medical treatment of humans, e.g., humans suffering from diseases that may be improved by transgene delivery. As shown in the examples, the transgene may be FIX or a variant thereof, such as the Padua mutant, but the transgene is by no means limiting, and additional transgenes may be contemplated in the present invention as described herein. In a 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 medical treatment of a human, wherein the anti-AAV5 antibody status is unknown (e.g., undetermined) and the human has not been subjected to medical treatment with an AAV5 gene therapy vector prior to the medical treatment. As stated, it may not be necessary to test for the presence of antibodies to 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 TAbs or NAbs in serum. Because it may not be necessary to test for TAbs or NAbs in serum, the size of the population eligible to receive treatment is greatly increased, and further, because it is not necessary to perform an NAb or TAb assay before treatment can begin, the greatly increased population size makes treatment and selection of eligible patients more convenient. All that may be required is to know whether the human patient has previously been subjected to AAV5 gene therapy treatment. It can be contemplated that prior treatment with AAV gene therapy treatment need not be limited to AAV5 alone but may also include treatment with other serotypes, such as serotype 8. For such subjects, it may be contemplated to include an NAb or TAb assay or test as described in the Examples section to confirm that NAb or TAb titers in the serum remain within the range observed in naive, untreated human patients.

[0027] In another embodiment, an AAV5 gene therapy vector is provided for use in medical treatment of a human, wherein the human has been pre-screened 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 to at most the 95th percentile of anti-AAV5 antibody levels observed in the human population. In this embodiment, human patients who may 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 the human population is from about 0 to about 1,000, or from about 1 to about 10,000.

[0028] The nth percentile herein is typically defined as the proportion (n%) of a human population within a distribution of 0% to n% that has an anti-AAV5 antibody level determined using an NAb assay or a TAb assay, such as those described in the Examples. For example, if human patients in a population (not 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 up to about 100% of the human population would be included, and therefore the anti-AAV5 antibody level detected in the entire population would be at most 10,000. It may be preferable to treat human patients with anti-AAV5 antibody levels at the 95th percentile, which corresponds to an NAb level determined using the assay described in the Examples of at most 4,500. It may be preferable to treat human patients if they have anti-AAV5 antibody levels at the highest 93rd or 90th percentile, which correspond to NAb levels determined using the assays described in the Examples of 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 highest 99th, 98th, 97th, 96th, 95th, 94th, 93rd, 92nd, 91st, 90th, 80th, or 70th percentile. Nevertheless, most, if not all, of the population can be expected to be eligible for treatment regardless of anti-AAV5 antibody titer. As shown in the Examples section, any anti-AAV5 antibody assay will suffice; i.e., either an NAb assay or a TAb assay or the like can be used to determine antibody titers in a human population to determine the 95th, 93rd, or 90th percentile. While the human population selected remains the same, the actual values ​​of the titers may vary (up to 10,000 for the NAb assay in the examples, or up to 5 for the TAb assay), because the observed titer values ​​are only relevant numbers when putting titers in perspective in the population.In any case, it is understood that the anti-AAV5 antibody titer levels determined in the population relate to a human population of at least 50 individuals as described in the Examples section.

[0029] Thus, in a further embodiment, an AAV5 gene therapy vector is provided for use in medical treatment of a human, wherein the human has been 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, as determined by an NAb ELISA assay described in the Examples, that corresponds to at most 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 they have an anti-AAV5 antibody level of at most the 99th, 98th, 97th, 96th, 95th, 94th, 93rd, 92nd, 91st, 90th, 80th, or 70th percentile.

[0030] In accordance with the present invention, it is understood that a subpopulation of the human population who would previously have been considered ineligible for treatment with AAV5 are now considered eligible for treatment with an AAV5 gene therapy vector, despite testing positive in an anti-AAV5 antibody assay. Thus, in a further embodiment, there is provided an AAV5 gene therapy vector for use in the medical treatment of a human, wherein the human tests positive for anti-AAV5 antibodies, and the human has not been previously treated with AAV5 or the like.

[0031] In a different embodiment, as shown in this embodiment that AAV5 antibody levels in endemic, untreated human populations enable efficient AAV5 gene therapy treatment, the present invention may also enable AAV gene therapy treatment, i.e., treatment of human patients subjected to AAV5. For example, means and methods for reducing the level of antibodies in the blood, thereby also reducing the level of anti-AAV5 antibodies, are known in the art. Such extracorporeal treatment of blood to remove antibodies from the blood may be employed to reduce the anti-AAV5 antibody titer in the blood to the same level observed with endemic exposure, i.e., in endemic, untreated human populations. 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). Thus, any method that can be employed to lower antibodies in the blood, including anti-AAV5 antibodies, can be utilized in the present invention to reduce anti-AAV5 antibody titers to such an extent that a human patient who was previously ineligible to receive AAV5-based gene therapy treatment because the human patient had previously been subjected to the treatment, can achieve anti-AAV5 antibody titers observed in endemic human populations and can be subjected to AAV5-based gene therapy.

[0032] In another embodiment of the present invention, the AAV5 gene therapy vector described above comprises at least 10 11 The AAV5 gene therapy vector is administered at a dosage equivalent to 10 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, at the dosages used, the concentration and / or amount of anti-AAV5 antibodies does not impair transduction. For example, to obtain a meaningful level of transgene expression, the amount of AAV5 gene therapy vector administered to a human patient in therapy far exceeds the anti-AAV5 antibodies present in the blood. From this perspective, there may be no upper limit. Nevertheless, the upper limit that can be considered is at most 10 16The dosage is in an amount equivalent to 10 capsids / kg body weight. It is understood that the dosage may be set in terms of dosage per patient or dosage per blood volume. 12 The dosage of 10 capsids / kg body weight is approximately 10 per patient, based on an average body weight of approximately 85 kg and an average blood volume of 5 L. 14 capsids or approximately 10 13 The dose is converted to capsids / L of patient blood volume. Therefore, whatever dose ranges are contemplated, these dose ranges can be easily recalculated based on these parameters. The dosage should be at least 1 x 10 12 At least 5 x 10 capsids / kg body weight 12 capsids / kg body weight, or at least 1 x 10 13 The dosage used in the Examples section is preferably equivalent to about 5×10 capsids / kg body weight. 13 capsids / kg body weight and approximately 2 x 10 14 AAV quantification of AAV capsid particle titers is readily determined and is well known in the art (see, inter alia, Kohlbrenner et al., Hum Gene Ther Meth. 2012 Jun; 23(3):198-203; Grimm et al., Gene Ther., 6(7):1322-1330, 1999).

[0033] The selected dosage may also be based on genome copies. Genome copies refer to the amount of vector genome contained in the AAV5 preparation. The gc titer of an AAV5 vector preparation can be easily determined by using qPCR to quantify the vector genome sequence. The AAV5 gene therapy vector should be at least 5 x 10 11 It is preferred to use a dosage equivalent to at least 5 x 10 g / kg body weight. 11 The dosage of capsids / kg body weight is approximately 5 x 10 per patient, based on an average body weight of approximately 85 kg and an average blood volume of 5 L. 12 gc or about 10 12gc / L of patient's blood volume. Therefore, whatever dose ranges are contemplated, these dose ranges can be easily recalculated based on these parameters. The selected dosage should be at least 1 x 10 12 gc / kg body weight, at least 2 × 10 12 gc / kg body weight, or 4 x 10 12 The dosage used in the Examples section may be about 5×10 12 gc / kg body weight and approximately 2 × 10 13 gc / kg body weight. There may be no upper limit, but the upper limit should be at most 10 15 It may be set to correspond to a dosage equivalent to gc / kg body weight.

[0034] As stated, the AAV5 gene therapy vectors according to the present invention are for use in medical treatment. The transgene contained in the AAV viral vector according to the present invention may not be a limitation of the present invention. Nevertheless, preferably and according to the example, the therapeutic gene encodes 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 may include variants of human factor IX, such as those described in WO2010029178, WO1999003496, WO2015086406, and WO2010012451, which are incorporated herein by reference in their entireties. In particular, it is shown in the Examples section that therapeutically meaningful quantities of protein can be obtained in human patients using FIX-encoding AAV5 vectors. Such proteins may be useful, for example, in the treatment of hemophilia A or hemophilia B.

[0035] Thus, in accordance with the present invention, 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, can achieve an amount of transgenic FIX protein in plasma ranging from about 0.02 micrograms / ml to up to about 5 μg / ml. Alternatively, when the AAV5 gene therapy vector is used in the treatment of hemophilia B patients with a severe phenotype, they may achieve 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 different plasma concentrations of FIX. In severe hemophilia B, plasma levels of FIX activity are below 1% of normal; in moderate forms, levels are 1% to 5%; and in mild forms, levels are 5 to 25% of normal. While healthy carrier individuals exist with intermediate FIX activity levels of 25% to 50% of normal, many carriers may have levels even greater than 50%.

[0036] Similarly, therapeutically effective amounts of other genes of interest can be expected to be well within the reach of one of ordinary skill in the art. Accordingly, the present invention is expected to be useful with any transgene. Additional suitable transgenes for delivery to a patient in a viral vector for gene therapy may be selected by one of ordinary skill in the art. These therapeutic nucleic acid sequences typically encode a product (e.g., a protein or RNA) for in vivo or ex vivo administration and expression in a patient to treat a genetic or non-genetic genetic defect, for example, by replacing or correcting a defect, to treat an epigenetic disorder or disease, or to treat a condition associated with dysregulation of a gene product. Such therapeutic genes desirable for performing 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 conductance 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 one of ordinary skill in the art to treat a particular disorder or disease. In a preferred embodiment, the rAAV vector comprises a transgene encoding a therapeutic protein or RNA, such as miRNA. The therapeutic protein can be factor IX (preferably human factor IX), factor VIII (preferably human factor VIII), lipoprotein lipase (LPL; e.g., LPL), or other therapeutic proteins. S447Xand variants thereof; see WO 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 preceding embodiments, in a preferred embodiment, the gene therapy is for treating, preventing, curing, and / or reversing a condition or disease, preferably a so-called orphan disease, which is understood herein to be a rare disease that is life-threatening, chronically debilitating, and / or inadequately treated and affects a small percentage of the population, for example, less than 1 in 1,500 people in the population. Typically, orphan diseases are genetic diseases and therefore lifelong, even if symptoms are not immediately apparent. In preferred embodiments, such conditions or diseases are lipoprotein lipase deficiency (LPLD), hemophilia B, acute intermittent porphyria (AIP), Sanfilippo B syndrome, Parkinson's disease (PD), congestive heart failure (CHF), hemophilia A, Huntington's disease, Duchenne muscular dystrophy (DMD), Leber's congenital amaurosis, X-linked severe combined immunodeficiency (SCID), adenosine deaminase-deficient severe combined immunodeficiency (ADA-SCID), adrenoleukodystrophy, chronic lymphocytic leukemia, acute lymphocytic leukemia, multiple myeloma, cystic fibrosis, sickle cell disease, hyperlipoproteinemia type 1, thalassemia, Alzheimer's disease, and the like. The disease is selected from the group consisting of 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 gangliosidosis, retinitis pigmentosa, homozygous familial hypercholesterolemia, lysosomal storage diseases, copper or iron storage disorders (e.g., Wilson's disease or Menkes disease), lysosomal acid lipase deficiency, hyperoxaluria, Gaucher's disease, Hurler's disease, adenosine deaminase deficiency, glycogen storage diseases, and retinal degenerative diseases (such as RPE65 deficiency and choroideremia).

[0038] In a further embodiment, the AAV5 gene therapy vector is for use in human medical treatment according to the present invention, and the use includes administration to the bloodstream, e.g., administration of the AAV5 gene therapy vector to the bloodstream. Blood may contain anti-AAV5 antibodies, and a delivery route via the bloodstream, e.g., via intravascular infusion or injection, is particularly contemplated. Delivery via the bloodstream allows delivery of the AAV5 vector to the target tissue. Such delivery to the target tissue may occur via systemic delivery. The present invention is not limited to administration to the bloodstream. Indeed, conventional and pharmaceutically acceptable routes of administration that may be contemplated include direct delivery to a target organ, tissue, or site (e.g., the liver or CNS), intranasal, intravenous, intramuscular, subcutaneous, intradermal, oral, and other parenteral routes of administration. However, a preferred target tissue that may be contemplated is the liver. Thus, it is most preferred that the AAV5 gene therapy vector delivers its transgene to the liver via the bloodstream. As stated, the AAV5 viral vector is administered in a sufficient amount to transfect the desired cells and provide sufficient levels of transduction and expression of the selected transgene to provide a therapeutic benefit without undue adverse effects or with a medically acceptable physiological effect, as can be determined by those skilled in the medical arts. Routes of administration may also be combined as needed. The dosage of the rAAV vector (i.e., AAV5 gene therapy vector) will primarily depend on factors such as the condition being treated, the selected gene, and the patient's age, weight, and health, and may therefore vary between patients.

[0039] AAV5 gene therapy vectors for use in human medical treatment according to the present invention are preferably AAV5 gene therapy vectors produced in insect cells. Without being bound 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 downside that rep and cap expression constructs are contained in the AAV capsid administered to patients, resulting in the transfer of only small amounts of the rep and cap expression constructs to the human subject. Expression of AAV rep and cap in human patients may be harmful from an immune perspective, especially in the case of human patients who may test positive for anti-AAV5 antibodies. Therefore, it may be preferable for AAV5 viral vectors to be administered to human patients to be produced in insect cells. Insect cell-based manufacturing is well established and includes, but is not limited to, the means and methods described in WO2007046703, WO2007148971, WO2009014445, WO2009104964, WO03042361, WO2008024998, WO2010114948, which are incorporated herein by reference.

[0040] In another embodiment, 1. A method for determining a human patient's eligibility to receive medical treatment with an AAV5 gene therapy vector, comprising: providing a serum sample from a human patient; determining the anti-AAV5 antibody titer; If the total anti-AAV5 antibody titer has a value 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 to receive medical treatment. A method is provided which includes:

[0041] Optionally, the method continues by: administering the AAV5 gene therapy vector to a qualified human patient. Includes:

[0042] It is understood that any of the meets and limitations described above with respect to embodiments related to medical uses of AAV5 gene therapy agents also apply to any of the methods described herein, e.g., for methods of delivery of or for determining eligibility for AAV5 gene therapy vectors. Preferably, the total anti-AAV5 antibody titer has a value within the range of 0.02 to 4, 0.02 to 3, or 0.02 to 2, as determined using the total anti-AAV5 antibodies (TAbs) described in the Examples.

[0043] In another embodiment, a method for determining a human patient eligible to receive medical treatment with an AAV5 gene therapy vector comprises: providing a serum sample from a human patient; determining the anti-AAV5 antibody titer; If the neutralizing anti-AAV5 antibody titer has a value 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 to receive medical treatment. Includes:

[0044] Optionally, the method continues by: administering the AAV5 gene therapy vector to a qualified human patient. Includes:

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

[0046] It is understood that the eligibility criteria described above are not the only criteria that can be used to select an AAV5 gene therapy treatment. Thus, if a human patient meets all other criteria, the eligibility of the human patient is determined by the anti-AAV5 antibody scale. In another embodiment, there is provided a method of treating a human in need thereof, comprising administering to said human an effective amount of an AAV5 gene therapy vector, the human has not been pre-screened using an assay to assess anti-AAV5 antibodies; Methods are provided wherein the human has not been subjected to medical treatment with an AAV5 gene therapy vector prior to the medical treatment.

[0047] In yet a further embodiment, there is provided a method of treating a human in need thereof, comprising administering to said human an effective amount of an AAV5 gene therapy vector, the human is pre-screened using an assay to determine anti-AAV5 antibodies; the human has not been subjected to medical treatment with an AAV5 gene therapy vector prior to the medical treatment; Methods are provided wherein the human has an anti-AAV5 antibody level that corresponds to at most the 95th percentile of anti-AAV5 antibody levels observed in the human population.

[0048] In another further embodiment, there is provided a method of gene delivery to a human in need thereof, comprising administering to said human an effective amount of an AAV5 gene therapy vector, the human is pre-screened using an assay to determine anti-AAV5 antibodies; the human has not been subjected to medical treatment with an AAV5 gene therapy vector prior to the medical treatment; Methods are provided wherein the human has an anti-AAV5 antibody level that corresponds to at most the 95th percentile of anti-AAV5 antibody levels observed in the human population.

[0049] In another embodiment, there is provided a method of gene delivery to a human in need thereof, comprising administering to said human an effective amount of an AAV5 gene therapy vector, the human has not been pre-screened using an assay to assess anti-AAV5 antibodies; Methods are provided wherein the human has not been subjected to medical treatment with an AAV5 gene therapy vector prior to the medical treatment.

[0050] Alternatively, or in combination with any one of the preceding embodiments, in a preferred embodiment, the AAV5 vector composition further comprises a pharmaceutically acceptable carrier, diluent, solubilizer, filler, preservative, and / or excipient. The rAAV vector carrying the therapeutic gene, preferably suspended in a biologically compatible solution or pharmaceutically acceptable delivery vehicle, can be administered to a patient. Suitable vehicles include sterile saline. Other aqueous and non-aqueous isotonic sterile injection solutions and aqueous and non-aqueous sterile suspensions known to be pharmaceutically acceptable carriers and familiar to those skilled in the art, may also be employed for this purpose. The viral vector is administered to a human patient in an amount sufficient as described above to transfect the desired cells and provide sufficient levels of transduction and expression of the selected transgene to provide a therapeutic benefit without undue adverse effects or with a medically acceptable physiological effect, as can be determined by one skilled in the medical arts. Further embodiments are as follows. [Embodiment 1] An AAV5 gene therapy vector for use in medical treatment of a human, wherein the human 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. [Embodiment 2] An AAV5 gene therapy vector for use in medical treatment of a human, wherein the human has been 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 to at most the 95th percentile of anti-AAV5 antibody levels observed in the human population. [Embodiment 3] 4. An AAV5 gene therapy vector for use in medical treatment of a human according to embodiment 3, wherein the human has tested positive for an anti-AAV5 antibody (body). [Embodiment 4] At least 10 12 4. 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 10 capsids / kg. [Embodiment 5] At least 10 12 5. 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] 6. An AAV5 gene therapy vector for use in human medical treatment according to any one of embodiments 1 to 5, for use in the treatment of a disease selected from the group consisting of hemophilia A or hemophilia B. [Embodiment 7] 7. An AAV5 gene therapy vector for use in human medical treatment according to any one of embodiments 1 to 6, encoding a FIX protein or a variant thereof for use in the treatment of hemophilia. [Embodiment 8] 8. An AAV5 gene therapy vector for use in human medical treatment according to any one of embodiments 1 to 7, wherein said use comprises administration into the bloodstream. [Embodiment 9] 9. An AAV gene therapy vector for use in human medical treatment according to any one of embodiments 1 to 8, wherein said use comprises delivery of said vector to the liver. [Embodiment 10] 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] 1. A method for determining a human patient's eligibility to receive medical treatment with an AAV5 gene therapy vector, comprising: providing a serum sample from a human patient; determining the anti-AAV5 antibody titer; If the total anti-AAV5 antibody titer has a value 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 to receive medical treatment. A method comprising: [Embodiment 12] 1. A method for determining a human patient's eligibility to receive medical treatment with an AAV5 gene therapy vector, comprising: providing a serum sample from a human patient; determining the anti-AAV5 antibody titer; If the anti-AAV5 antibody titer has a value within the range of 3 to 5,000 as determined using a neutralizing anti-AAV5 antibody assay as determined by the NAb assay described in the Examples, the patient may be considered eligible to receive medical treatment. A method comprising: [Example]

[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 had ≥ 4 bleeds per year or hemophilic arthropathy. Further details of the study can be found on the NIH clinicaltrials.gov website (NCT02396342). The study was approved by the Institutional Review Board / Institutional Review Board 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] An AAV5 vector incorporating a codon-optimized wild-type hFIX gene 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) was used in the study. The vector was produced using a baculovirus expression system in accordance with Good Manufacturing Practices for Drugs and Quasi-drugs. The vector genome copy titer (gc) was determined using qPCR. The capsid:gc ratio was approximately 10, i.e., 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 an SEC column, which is selected for its ability to separate AAV particles from smaller matrix components. In this method, a calibration curve is generated using an AAV vector preparation with known total particle concentration. In the calibration curve, the amount of total particles injected is plotted 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 successive ascending dose cohorts: Cohort 1 (n=5, participants 1-5) was administered 5 x 10 12 Cohort 2 (n = 5, participants 6–10) received 2 × 10 13 Cohort 1 consisted of adult males with a mean age of 69 years (range, 35-72 years) and a mean body weight of 84.5 kg (range, 71.2-89.1 kg). Cohort 2 consisted of adult males with a mean age of 35 years (range, 33-46 years) and a mean body weight of 84.0 kg (range, 71.4-96.0 kg). Efficacy outcome measures included FIX plasma activity measurements. In addition, serum from subjects was obtained for neutralizing AAV5 antibody titer (NAb titer) and total AAV5 antibody titer (TAb) analysis.

[0053] Control sera from healthy donors were commercially obtained from SeraLab (West Sussex, UK). All information provided relating to these sera is listed below.

[0054] [Table 1]

[0055] Neutralizing AAV5 antibody (NAb) titer Measurement of NAbs in human serum was assessed based on a sensitive in vitro assay using AAV5 carrying the transgene luciferase (AAV5-luc) 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 with phenol red (Gibco, ref. #31966) / 10% FBS (Greiner, ref. #758093) / 1% PenStrep (Gibco, ref. #15140) Phenol red-free DMEM (Gibco, ref. #21063) / 1% Pen-Strep (Gibco, ref. #15140) 1x PBS- / - (Gibco, reference #14190) 1x Trypsin EDTA (Gibco, Reference #25200) Poly-L-lysine (PLL) solution (2.5%) (Sigma-Aldrich, reference #8920-100) 96-well flat-bottom black culture plates (Costar, ref. #3916) Clear 96-well flat-bottom plate (Corning, reference #3596) ONE-Glo Luciferase Assay System (Promega, Ref. #E6120) Glo Lysis Buffer, 1x (Promega, Ref. #E2661) AAV5-CMV-luc (e.g., using AAV5-CMV-73QlucHtt from PKO, titer: 4e13gc / ml)

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

[0058] The next day, serial dilutions of plasma were prepared in medium (DMEM / 1% PS, phenol red-free / 10% FBS) in clear 96-well plates. After virus addition (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 wells designated A2 through A11 (negative control wells) and 70 μl of medium 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 through H11 (positive controls).

[0060] Plasma samples were added to wells B2, C2, D2, E2, F2, and G2 (140 μl / well), resulting in the first dilution: 1. Consequently, serial dilutions of plasma were performed across the plate by transferring 70 μl from column 2 to column 3 (dilution 2), 3 to 4 (4), 4 to 5 (8), 5 to 6 (16), 6 to 7 (32), 7 to 8 (64), 8 to 9 (128), 9 to 10 (256), and 10 to 11 (512), followed by discarding 70 μl from column 11. AAV5-CMV-73QlucHtt was diluted to 6 × 10 in medium (DMEM / 1% PS, phenol red-free / 10% FBS). 9 Prepare 70 μl / well of 6 x 10 9Add the AAV5(160)-CMV-73QlucHtt virus dilutions at 1000µg / ml to the plasma dilution plate, except for wells A2 through A11 (negative control). Carefully place the plate on 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 day before and replaced with 100 μl / well of the prepared plasma dilutions by pipetting them from the clear plate into the black plate containing Hek293T cells. 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 1x PBS (100 μl / well), after which 100 μl / well of Glo Lysis Buffer was added to the plate and incubated at room temperature for 5 minutes to allow lysis. Following this, 100 μl / well of the reagent from the ONE-Glo Luciferase Assay System was added (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 subtraction of background activity, the percent neutralization for each serum dilution is calculated, and anti-AAV5 neutralizing antibody titers are determined using LabKey software analysis, which fits a curve to the neutralization profile. LabKey then uses this curve to calculate neutralizing antibody titers for a selected benchmark, the area under the curve (AUC), and an error estimate. A four-parameter method was used to calculate the curve fit. LabKey calculates the IC50, the dilution at which the antibody inhibits transduction by 50%. LabKey also calculates "point-based" titers 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 either side of the target neutralization percentage. Each run included a positive control (wells with AAV5-LUC but no sample serum), a negative control (wells with medium only, no sample serum, no AAV5-LUC), and a negative control sample serum (heat-inactivated FBS) to assess the specificity of AAV5-LUC neutralization. FBS should not have anti-AAV5 neutralizing properties when measured as a sample.

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

[0063] Results and Discussion All human patients in both cohorts exhibited significant improvements in FIX activity, with most patients improving by a change in phenotype from severe to mild (Table 2), resulting in a substantial reduction or even absence of the use of prophylactic administration of FIX protein. Variation was observed between the observed FIX activity levels between patients and between cohorts. Variation in FIX activity levels did not correlate with the NAb or TAb status of the human patients.

[0064] The previously reported prevalence of TAbs against AAV5 (40%, Boutin et al., Hum Gene Ther. 2010 Jun;21(6):704-712) was generally consistent with the results of this analysis (30%). Results obtained using a luciferase-based NAb assay (see Figures 1 and 2) suggest a similar prevalence of AAV5 (neutralizing) antibodies, with positive signals recovered for 14 of 50 screened control sera (28%), a rate consistent with a recent study (Li C et al., Gene Ther. 2012 Mar;19(3):288-94). Results from sera obtained from human patients prior to gene therapy treatment were also consistent with a recent study, with 3 of 10 sera found to be positive in both the NAb and TAb assays (30%). Total antibodies assessed by ELISA and neutralizing antibodies assessed by the luciferase-based assay correlated closely, suggesting that both assays detect the same entity (see Figure 2A).

[0065] Additionally, the presence of neutralizing antibody titers after treatment was also tested, with approximately 10 6 The antibody titers found in endemic, untreated humans are therefore significantly different from 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 a rise in IgG, typical of initial exposure to antigen. Furthermore, there was no evidence of ALT (alanine aminotransferase) elevation or capsid-specific T cell activation in treated patients with pre-existing NAbs. Thus, administration of AAV5-based gene therapy in patients with endemic, pre-existing NAbs was well tolerated, without ALT elevation or T cell activation.

[0066] In conclusion, the presence of anti-AAV5 antibodies detected in vitro by either the NAb or TAB assay did not predict or indicate impaired in vivo transduction. There was no clear correlation between the presence of NAb pretherapy and posttherapy FIX levels resulting from AAV5 FIX gene transfer. Notably, the highest responders in Cohort 1, who received a lower dose of AAV5 vector, also had the highest levels of NAb and TAb antibodies detected. The range of anti-AAV5 titers observed in the healthy population indicates that antibody levels in a healthy population not subjected to AAV5 gene therapy treatment do not impair AAV5 transduction in vivo. This is because the highest titer observed in the healthy population was close in range to the highest titer observed in Patient 5 of Cohort 1. Therefore, it appears feasible to eliminate the need to test for the presence of anti-AAV5 antibodies in untreated populations prior to treatment with AAV5 gene therapy vectors.

[0067] [Table 2]

Claims

1. 1. A pharmaceutical composition comprising an AAV5 gene therapy vector for use in human medical treatment, comprising: the human is pre-screened using an assay to determine anti-AAV5 antibodies; the human has not been subjected to medical treatment with an AAV5 gene therapy vector prior to the medical treatment; the human has an anti-AAV5 antibody level that corresponds to less than or equal to the 95th percentile of anti-AAV5 antibody levels observed in a healthy population of humans not subjected to medical treatment with an AAV5 gene therapy vector; The pharmaceutical composition, wherein the human has tested positive for anti-AAV5 antibodies.

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

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

4. The pharmaceutical composition according to any one of claims 1 to 3, which is used in the treatment of a disease selected from the group consisting of hemophilia A and hemophilia B.

5. The pharmaceutical composition of any one of claims 1 to 4, for use in the treatment of hemophilia, wherein the AAV5 gene therapy vector encodes a FIX protein or a variant thereof.

6. The pharmaceutical composition of any one of claims 1 to 5, wherein said use comprises administration to the bloodstream.

7. The pharmaceutical composition of any one of claims 1 to 6, wherein said use comprises delivery of said AAV5 gene therapy vector to the liver.

8. 1. A method for determining a human patient's eligibility to receive medical treatment with an AAV5 gene therapy vector, comprising: Providing a serum sample from a human patient; determining the anti-AAV5 antibody titer of the serum sample; wherein the human patient is considered eligible to receive the medical treatment if the anti-AAV5 antibody titer has an anti-AAV5 antibody level that corresponds to the 95th percentile or less of anti-AAV5 antibody levels observed in a healthy human population not subjected to medical treatment with an AAV5 gene therapy vector and tests positive for anti-AAV5 antibodies.

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