EA044191B1 - MEANS AND METHODS FOR AAV GENOTHERAPY IN HUMANS - Google Patents

Description

Field of technology to which the invention relates

The present invention relates to agents 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 directed against the AAV intended for use in the specified treatment.

BACKGROUND OF THE INVENTION

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

One of the major challenges for successful AAV vector administration is overcoming the presence of neutralizing antibodies (immunoglobulins) (NAbs) that arise after exposure to wild-type AAV or AAV-based vectors. In both cases, neutralizing serotype-specific antibodies directed against viral capsid proteins may reduce the efficiency of gene transfer using AAVs of the same serotype.

Relatively lower titers of endemic NAB were observed for human serotype AAV5 compared with other serotypes (Boutin et al. Hum Gene Ther 2010, 21:704-712). It has been published that when treating humans with AAV, already such low titers of endemic NAB affect transduction and lead to greatly reduced transgene expression (Manno et al., Nature Medicine, 2006, 12(3), 342-347). Thus, the general consensus in the field is to avoid treating patients with cumulative NAB titers. Thus, current clinical practice in relation to pre-existing immunity involves screening human patients to appropriately exclude patients who 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 tried to reduce NAb production on the first administration to allow a second administration (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)). In addition, methods have been proposed to overcome pre-existing antibodies, including plasma exchange and the use of immunosuppressive regimens (for example, 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 methods have been tested in animal models with limited success.

Thus, there is a need in the art to enable rAAV gene therapy vectors to be administered to human patients who have or are suspected of having AAV neutralizing antibodies.

Brief description of the invention

The inventors of the present invention have now unexpectedly discovered that, in particular for AAV5 gene therapy vectors, and, contrary to assumptions in the prior art, human patients having endemic pre-existing anti-AAV5 antibodies (i.e., pre-existing anti-AAV5 antibodies, resulting from endemic exposure) may be considered suitable for treatment. This differs from the view in the prior art that the presence of neutralizing antibodies should be considered as an exclusion criterion, for example, for patients participating in clinical trials. In other words, the view in the prior art is that patients having anti-AAV5 antibodies, more specifically, endemic pre-existing anti-AAV5 antibodies, are not considered suitable for treatment using an AAV5 gene therapy vector. The unexpected finding described herein that human patients having endemic pre-existing anti-AAV5 antibodies may be considered eligible for treatment is not limited to the subpopulation of human patients, for example, found to have very low levels of pre-existing anti-AAV5 antibodies, but instead, it is found to apply substantially to most, if not all, patients in a population of people who are assessed as positive for pre-existing anti-AAV5 antibodies and who have not previously received any AAV5 gene therapy.

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 is not prescreened using an anti-AAV5 antibody assay and wherein the individual has not been subjected to medical treatment using a gene therapy AAV5 vector prior to indicated medical treatment. In other words, the present invention

- 1 044191 relates to an AAV5 gene therapy vector for use in the medical treatment of a human where the anti-AAV5 antibody status is unknown and where said human has not been medically treated using an AAV5 gene therapy vector prior to said medical treatment. In accordance with one aspect, the present invention may enable the treatment of patients or the inclusion in clinical trials of patients who have not been previously treated with an AAV5 gene therapy vector without screening for the presence of anti-AAV5 antibodies prior to medical treatment.

In a further aspect, the present invention relates to an AAV5 gene therapy vector for use in the medical treatment of a human, wherein said human is prescreened using an anti-AAV5 antibody assay, and wherein said human has not been medically treated with an AAV5 gene therapy vector prior to said medical treatment, wherein said individual has an anti-AAV5 antibody level corresponding to at most the ^100th percentile, preferably at most the 95th percentile, of anti-AAV5 antibody levels as observed in the human population. Preferably, said human patient is tested positive for anti-AAV5 antibodies.

Description of the figures

Fig. 1. Nab analysis results for pre-treatment samples. A: neutralization results of ten samples after pre-dosing. The fifty percent mark is indicated by a dotted line. B: Curve fitting results for three positive samples 3, 4, 5. A four-parameter curve was fitted using nonlinear regression. Titers were calculated as the theoretical dilution at which the fitted curve passed the 50% mark (shown above the horizontal axis).

Fig. 2A. AAV5 neutralizing antibodies versus total anti-AAV5 antibodies. Neutralizing antibody (NAb) titer compared with total antibody (TAb) ELISA results as published 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 superimposed (A, for study subjects 3, 4, and 5, as indicated).

Fig. 2B. Anti-AAV5 NAb titer vs. FIX levels. The percentage of FIX activity in cohort 1 after dosing is plotted against the pre-dosing NAb titer.

Fig. 3. Nab versus AAV titer scale. Human population percentiles (50 subjects) for anti-AAV5 NAb titers are shown. It should be noted that the Nab titers observed in the human population are well beyond the Nab titers observed in AAV5-treated human patients.

Fig. 4. The amino acid sequence of wild-type AAV5 VP1 is shown. The starting amino acid positions of VP2 (T, due to the ACG start site) and VP3 (M) are underlined.

Fig. 5. Shown is the VP1 amino acid sequence of a hybrid VP1 sequence consisting of the N-terminus of the AAV2-derived VP1 sequence (underlined) linked to the AAV5-derived VP2 and VP3 coding sequence. The VP1 protein is thus an AAV2/AAV5 fusion capsid protein. The expression constructs used for AAV capsids encoding the VP1 fusion may also encode VP2 and VP3 sequences, which may not be the VP2 and VP3 fusion capsid proteins, but rather the wild-type AAV5 VP2 and VP3 protein sequence.

Fig. 6. Shown is the amino acid sequence of wild-type AAV5 VP1 having an Ala insertion between positions 1 and 2 of the wild-type AAV5 sequence. Thus, the VP1 capsid consists of the wild-type AAV5 sequence with an inserted amino acid, and the encoded proteins VP2 and VP3 are the wild-type AAV5 VP2 and VP3 proteins without modification.

Definitions

AAV vector refers to a recombinant adeno-associated virus (AAV) vector derived from wild-type AAV using molecular methods. An AAV vector differs from a wild-type (wt) AAV vector because at least a portion of the viral genome is replaced by a transgene that is a non-natural nucleic acid relative to the wild-type AAV nucleic acid sequence.

An AAV vector comprising combinations of an AAV capsid and an AAV genome ITR can be produced using methods known in the art, as 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), the contents of which are incorporated herein by reference. Alternatively, AAV vectors can be produced in insect cells using the baculovirus expression system (BEVS). The original baculovirus system for producing rAAV is described in Urabe et al. (Urabe et al. [2002] Human Gene Therapy 13(16): 1935-1943) and consists of three baculoviruses, namely, Bac-Rep, Bac-cap and Bac- vec, co-infection of which into insect cells with, for example, SF9 resulted in the production of rAAV. The properties of the rAAV thus obtained, ie, physical and molecular characteristics, including activity, were not significantly different from rAAV produced in mammalian cells (Urabe [2002], supra). The original baculovirus system from

- 2 044191

Urabe (2002, above) developed further (see, for example, 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).

The term transgene is used to refer to a non-naturally occurring nucleic acid, as applied to the AAV nucleic acid sequence. It is used to refer to a polynucleotide that can be introduced into a cell or organism. Transgenes include any polynucleotide, such as a gene that encodes a polypeptide or protein, a polynucleotide that is transcribed into an inhibitory polynucleotide, or a polynucleotide that is not transcribed (eg, lacking an expression control element 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 containing an expression regulatory element, such as a promoter or transcription regulatory sequence, operably linked to a coding sequence and a 3' termination sequence.

Transduction refers to the transfer of a transgene into a recipient host cell via a viral vector. Transduction of a target cell with the rAAV vector of the invention results in the transfer of the transgene contained in the vector into the transduced cell. Host cell or target cells refers to the cell into which the DNA is delivered, such as, for example, synoviocytes or synovial cells of an individual. AAV vectors are capable of transducing both dividing and non-dividing cells.

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

Sequence identity and sequence similarity can be determined by aligning two peptides or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar length are preferably aligned using a global alignment algorithm (eg, Needleman-Wunsch), which aligns sequences optimally over their entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (eg, SmithWaterman). Sequences can then be designated as substantially identical or substantially similar when they (if optimally aligned, such as through the GAP or BESTFIT programs using default parameters) share at least a certain minimum percentage of sequence identity (as defined below). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their full length, maximizing the number of matches and minimizing the number of gaps. Global alignment is suitably used to determine sequence identity when two sequences are of similar length. Typically, default GAP parameters are used, with gap opening penalty=50 (nucleotides)/8 (proteins) and gap continuation penalty=3 (nucleotides)/2 (proteins). For nucleotides, the default scoring matrix is nwsgapdna, and for proteins, the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and percentage sequence identity scores can be determined using computer programs such as GCG Wisconsin, version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open source software code such as the needle program (using the global Needleman-Wunsch algorithm) or water (using the local Smith-Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as for GAP above, or using the default settings (for both needle and water, and for both protein and DNA alignments, the default skip penalty is 10.0, and the default skip penalty is 0.5; the default score matrices are Blossum62 for proteins and DNAFull for DNA). When sequences have significantly different overall lengths, local alignments, such as using the Smith-Waterman algorithm, are preferred. Alternatively, the percentage of similarity or identity can be determined by searching public databases using algorithms such as FASTA, BLAST, etc.

As used herein, gene therapy is the insertion of nucleic acid sequences (eg, a transgene as defined herein) into cells and/or tissues of an individual to treat a disease. A transgene may be a functional mutant allele that replaces or complements a defective allele. Gene therapy also includes the insertion of a transgene that is inhibitory in nature, i.e., that inhibits, reduces, or reduces the expression, activity, or function of an endogenous gene or protein, such as an unwanted or aberrant (eg, pathogenic) gene or protein. Such transgenes may be exogenous. By exogenous molecule or sequence is meant a molecule or sequence not normally found in the cell, tissue and/or individual being treated. Both acquired and hereditary diseases are amenable to gene therapy. The AAV5 gene therapy vector thus refers to an AAV5 vector for gene therapy.

In this specification and claims, the word contain and its conjugations are used in their non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.

In addition, reference to an unspecified singular element does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that one and only one of the elements be present. An unspecified singular element thus usually means at least one.

The word approximately or about, when used in association with a numerical value (about 10, about 10), preferably means that the value may be a given value of 10, and more or less than 10% of that value.

Detailed Description of the Invention

As indicated, it has been surprisingly discovered that, particularly for AAV5 gene therapy vectors, human patients having endemic pre-existing anti-AAV5 antibodies may be considered eligible for treatment. This differs from the general consensus that pre-existing AAV antibodies against a particular serotype preclude gene therapy treatment using that serotype. Without being bound by theory, AAV5 may be a serotype for which endemic pre-existing anti-AAV5 antibody titers, as found in the human population, are relatively low compared to other serotypes. This may occur due to the route of infection, which may differ between serotypes, and/or may occur in the presence or absence of co-infection with a helper virus. Additionally, AAV5 is the most distinct and phylogenetically distinct from other primate AAV serotypes, which may also contribute. Whatever the basis of the present invention, it allows most, if not all, human populations to be suitable for treatment using gene therapies based on the AAV5 serotype or the like. This includes the subset of the population of people who are negative for anti-AAV5 antibodies, as well as the subset of the population of people found to be positive for anti-AAV5 antibodies, not including the (currently) very small subset of the population of people who have been treated with AAV5 gene therapy or etc. Human populations that have been treated with AAV5 gene therapy exhibit such high titers of anti-AAV5 antibodies (approximately 10 ⁴ or more compared to endemic AAV5-infected individuals) that they are considered unsuitable for treatment using AAV5 gene therapy vectors or the like.

Thus, in a first aspect, the present invention provides an AAV5 gene therapy vector for use in the medical treatment of a human, wherein said human has not been prescreened using an anti-AAV5 antibody assay and wherein said human has not been medically treated with an AAV5 gene therapy vector prior to specified medical treatment.

The complete genome of AAV5 and other AAV serotypes has been sequenced (Chiorini et al. 1999, J. of Virology Vol. 73, No. 2, p1309-1319), and the nucleotide sequence is available in GenBank (Accession No. AF085716; February 23, 2015 ). It is understood that gene therapy vectors based on wild-type AAV5 contain at least AAV5 capsid proteins containing capsid proteins VP1, VP2 and VP3 corresponding to or at least substantially identical to the indicated amino acid sequences. Substantially identical thereto includes having at least 80%, at least 85%, at least 90%, or at least 95% amino acid sequence identity with them. The AAV5 capsid VP1 protein sequence, against which sequence identity can be determined, is shown in FIG. 4. Such sequences may be naturally occurring AAV viral sequences falling phylogenetically within the AAV5 clade (eg, as shown in FIG. 4).

Serotype is traditionally determined based on the lack of cross-reactivity between antibodies against one virus compared to another virus. Such cross-reactivity differences are typically due to differences in capsid protein sequence/antigenic determinants (eg, due to sequence differences in VP1, VP2, and/or VP3 of AAV serotypes). By traditional definition, serotype means that the virus of interest

- 4 044191 was tested against sera specific for all existing and characterized serotypes for neutralizing activity and for detecting antibodies neutralizing the virus of interest. As more natural isolates of the virus are discovered and capsid mutants are developed, serological differences from any currently extant serotype may or may not be present. For convenience, AAV5 serotypes include AAVs with capsid sequence modifications that are not characterized as being a distinct serotype, which may also constitute a subgroup or variant of the AAV5 serotype. Such variants typically have significant sequence identity.

Non-natural capsid sequences may also be provided in accordance with the invention, for example, amino acid sequences exposed to the serum (exposed to the outer space) may be from one serotype, while non-exposed amino acid sequences within the capsid may be from other serotypes and/or allow greater variety. Since the crystal structure of AAV5 (e.g. Govindasamy et al. J. Virol. Oct. 2013, vol. 87 no. 20; 11187-11199) is known, sequences that are not exposed and, for example, are in the interior of the AAV5 capsid, may be replaced by sequences from other serotypes and/or allow greater sequence variability. For example, the amino acid sequence of VP1, not contained in VP2 and VP3, is located in the internal part. This sequence may be derived, for example, from serotype 2, while the amino acid sequences of VP2 and VP3 may be based entirely on AAV5 (see, for example, FIG. 5). Such an AAV5 gene therapy vector capsid is, in terms of serotype and, in terms of neutralizing antibody, indistinguishable from a completely wild-type capsid (see, inter alia, WO2000028004 and Urabe et al. J Virol, Feb. 2006, Vol. 80, No. 4 p. 1874-1885). Such non-natural capsid sequences are hybrid sequences, and it is understood that such hybrid vectors are also the AAV5 gene therapy vectors of the invention. In addition, AAV5 capsid sequences may also have one or more amino acids inserted or replaced to improve vector production and/or activity, such as, but not limited to, those described in WO2015137802 and as shown, for example, in FIG. 6. Such AAV5 capsids with minor modifications can also be considered as serotype AAV5.

It will be understood that the AAV5 vectors or AAV5 gene therapy vectors of the invention refer to an AAV5 vector capsid comprising a vector genome having a gene of interest contained between AAV inverted repeats, which may, but not necessarily, be an AAV5 ITR. Thus, the AAV5 vectors of the invention are delivery vehicles designed to deliver their load, a vector genome having a transgene, for example a transgene intended to provide a benefit to a human, to its target cells, for example liver cells or cardiac muscle cells . Thus, AAV5 vectors can be considered useful in the medical treatment of a human, for example, a human patient suffering from a disease that can be alleviated by delivery of a transgene. As shown in the examples, the transgene may be FIX, or variants thereof, such as the Padua mutant, but the transgene is not limited in any way and additional transgenes may be provided by the invention as described herein. Also, in one further embodiment, the human patient may be a male human patient.

In another embodiment, the present invention provides an AAV5 gene therapy vector for use in the medical treatment of a human where the anti-AAV5 antibody status is unknown (e.g., has not been determined), and where said human has not been medically treated using an AAV5 gene therapy vector prior to said medical treatment . As indicated, it may not be necessary to test such a patient for the existence of anti-AAV5 antibodies. As shown in the Examples section, it has been shown that about 30% of the human population is positive for the presence of TAbs or NAbs in serum. Since TAb or NAb testing in serum may not be necessary, the size of the population available for treatment is greatly increased, furthermore, it makes treatment and selection of eligible patients more convenient since there is no need to perform NAb or Tab testing before treatment can be started . All that may be necessary is to know whether or not the human patient has previously undergone AAV5 gene therapy. It may be contemplated that prior treatment using AAV gene therapy may not be limited to AAV5 alone, but may include treatment using other serotypes as well, such as serotype 8. For such subjects, NAb or Tab testing or testing, such as described in the Examples section to confirm that the serum NAb or TAb titer remains within the range observed in naïve untreated human patients.

In another embodiment, the present invention provides an AAV5 gene therapy vector for use in the medical treatment of a human, wherein the human is prescreened using an anti-AAV5 antibody assay and wherein the human has not been medically treated using the gene therapy.

- 5 044191 torus AAV5 before specified medical treatment, wherein the specified individual has a level of anti-AAV5 antibody corresponding to at most the 95th percentile of anti-AAV5 antibody levels as observed in a human population. In this embodiment, a human patient who may benefit from gene therapy treatment is prescreened using an anti-AAV5 antibody assay. As shown in the example section, the range of anti-AAV5 antibody levels observed in a human population, i.e., the observed range of anti-AAV5 antibody levels is from about 0 or about 1, to about 10,000.

The nth percentile as used herein is generally defined as the proportion of a population of people, n%, falling within the distribution of 0% to n% having levels of anti-AAV5 antibody, as determined using a NAb assay or a Tab assay, such as as described in the examples. For example, when a human patient in a population (not previously treated with an AAV5 vector) has a level of anti-AAV5 antibody as determined using a Nab assay as described in the Examples, the level of anti-AAV5 antibody detected in the entire population should be at most 10,000 because this specifies that up to approximately 100% of the human population may be included. It may be preferable to treat a human patient with a level of anti-AAV5 antibody up to the 95th percentile, which corresponds to a Nab level as determined using the assay as described in the examples, at most 4500. It may be preferable to treat a human patient with a level of anti-AAV5 antibodies up to the 93rd percentile or 90th percentile, which corresponds to a Nab level as determined using the assay as described in the Examples, at most 3000 or 1000, respectively (see FIG. 3). In another embodiment, it may be preferable to treat a human patient with anti-AAV5 antibody levels of up to 99, 98, 97, 96, 95, 94, 93, 92nd, 91st, 90th, 80th or 70th percentile. However, it can be assumed that most, if not all, of the population is suitable for 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 the like, can be used to determine antibody titers in a human population to determine the 95th percentile, 93- th percentile or 90th percentile. The population of people selected remains the same, while the actual titer values may vary (up to 10,000 for the example NAb assay or up to 5 for the TAb assay). This is because observed titer values are simply numbers that are meaningful only when considered in terms of titers in a population. It is understood that in any case, the levels of anti-AAV5 antibody titers, as determined in the population, refer to a population of at least 50 people, as described in the examples section.

Thus, in a further embodiment, the present invention provides an AAV5 gene therapy vector for use in the medical treatment of a human, wherein the human is prescreened using an anti-AAV5 antibody assay, and wherein the human has not been medically treated with the AAV5 gene therapy vector prior to of said medical treatment, wherein said individual has a level of anti-AAV5 antibody, as determined by the Nab ELISA assay as described in the Examples, corresponding to at most the 95th percentile of anti-AAV5 antibody levels as observed in the human population. In another embodiment, it may be preferable to treat a human patient with an anti-AAV5 antibody level of up to 99, 98, 97, 96, 95, 94, 93, 92nd, 91st, 90th, 80th or 70th percentile.

It is understood that, according to the invention, a subpopulation of the human population that was not previously considered suitable for treatment using AAV5 may now be considered suitable for treatment using an AAV5 gene therapy vector, rather than being tested as positive in an anti-AAV5 antibody assay. Thus, in a further embodiment, the present invention provides an AAV5 gene therapy vector for use in the medical treatment of a human, wherein the human has tested positive for anti-AAV5 antibodies, and wherein the human has not previously been treated with AAV5 or the like.

In various embodiments, since the present embodiment shows that anti-AAV5 antibody levels in an endemic, untreated human population allow effective gene therapy treatment using AAV5, the present invention may also allow treatment of human patients who have been subjected to gene therapy treatment using AAV, i.e. AAV5. For example, agents and methods are known in the art that can reduce antibody levels in the blood and thus also reduce anti-AAV5 antibody levels. Such in vitro blood treatments, where antibodies are removed from the blood, can be used to reduce anti-AAV5 antibody titers in the blood to achieve the same levels as observed from endemic exposure, ie, 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). Thus, any method that can be used to reduce levels of antibodies in the blood, including anti-AAV5 antibodies, can be used according to the invention to reduce the titer of an anti-AAV5 antibody to such an extent that it would not previously be suitable for human patients. for treatment because they have previously been subjected to AAV5-based gene therapy, obtain anti-AAV5 antibody titers as observed in an endemic human population, and allow them to undergo AAV5-based gene therapy.

In another aspect of the invention, said AAV5 gene therapy vector, as described above, is administered at a dose corresponding to at least 10 ¹¹ capsids/kg body weight. It is understood that the observation made by the present inventors regarding the presence of anti-AAV5 antibodies may be dose dependent. In other words, at the doses used, the concentration and/or amount of anti-AAV5 antibodies does not impair transduction. The amount of AAV5 gene therapy vector administered to a human patient during treatment to obtain, for example, a significant level of transgene expression, significantly exceeds the levels of anti-AAV5 antibodies present in the blood. In this regard, there is no need to consider an upper limit. However, the upper limit that can be considered is a dose corresponding to at most 10 ¹⁶ capsids/kg body weight. It will be understood that doses can be set as dose per patient or dose per blood volume. A dose of at least 10 ¹² capsids/kg body weight translates to approximately 10 ¹⁴ capsids per patient or approximately 10 ¹³ capsids/L patient blood volume, based on an average body weight of approximately 85 kg and an average blood volume of 5 L. Thus, whatever dosage range is provided can be easily recalculated based on these parameters. Preferably, the dose corresponds to at least 1 x 10 ¹² capsids/kg body weight, at least 5 x 10 ¹² capsids/kg body weight or at least 1 x 10 ¹³ capsids/kg body weight. The dose used in the Examples section is approximately 5 x 10 ¹³ capsids/kg body weight and approximately 2 x 10 ¹⁴ capsids/kg body weight. AAV quantification of AAV capsid particle titers is easily determined and well known in the art (among others, Kohlbrenner et al., Hum Gene Ther Meth. June 2012, Vol. 23, No. 3: 198-203; Grimm et al., Gene Ther., Vol. 6, No. 7, p, 1322-1330, 1999).

The selected dose may also be based on genomic copies. Genomic copies indicate the number of vector genomes contained in an AAV5 preparation. The HA titer for an AAV5 vector preparation can be easily determined using qPCR to quantify the genomic sequence of the vector. Preferably, said AAV5 gene therapy vector is used at a dose corresponding to at least 5 x 10 ^pg /kg body weight. A dose of at least 5 x 10 ^p capsids/kg body weight translates to approximately 5 x 10 ¹² gc per patient or approximately 10 ¹² gc/L patient blood volume, based on an average body weight of approximately 85 kg and an average blood volume of 5 L. Thus, whatever dosage range is provided can be easily recalculated based on these parameters. The selected dose may be at least 1 x 10 ¹² gc/kg body weight, at least 2 x 10 ¹² gc/kg body weight, or 4 x 10 ¹² gc/kg body weight. The dose used in the Examples section is approximately 5 x 10 ¹² gc/kg body weight and approximately 2 x 10 ¹³ gc/kg body weight. Although there may be no upper limit, it can be set to correspond to a dose corresponding to at most 10 ¹⁵ gk/kg body weight.

As stated, the AAV5 gene therapy vector of the invention is intended for use in medical treatment. The transgene contained in the AAV viral vector of the invention may not be a limitation of this invention. However, preferably, and in accordance with the examples, 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. B NEngl J Med 2014; 371(21): 1994-200, and may include variations thereof such as those described in WO 2010029178, WO 1999003496, WO 2015086406 and WO 2010012451, the entire contents of which are incorporated herein by reference. In particular, the Examples section demonstrates that therapeutically significant amounts of protein can be produced using AAV5 FIX-encoding vectors in human patients. They can be used, for example, in the treatment of hemophilia A or hemophilia B.

Thus, accordingly, for an AAV5 gene therapy vector for use in human medical treatment of the invention, wherein said AAV5 gene therapy vector is used for the treatment of hemophilia B, the amount of transgenic FIX protein produced in plasma may range between about 0.02 μg/ml and up to approximately 5 μg/ml. Alternatively, when the AAV5 gene therapy vector is used in the treatment of hemophilia B patients having a severe phenotype, a moderate or mild phenotype, or even a phenotype as observed in healthy individuals, is obtained after treatment. Hemophilia B can be classified into three classes, each characterized by the presence of different concentrations of FIX in plasma. In severe hemophilia B, plasma FIX activity levels are below 1% of normal; in moderate form, levels are between 1% and 5%; in mild forms, between 5 and 25% of normal levels. There are healthy carrier individuals who have average levels of FIX activity between 25% and 50% of normal, but many carriers may have levels even exceeding 50%.

Likewise, therapeutically effective amounts of other genes of interest can be expected to be within the reach of one skilled in the art. Thus, it is contemplated that the invention can be used with any transgene. Additional

- 7 044191 suitable transgenes to be delivered to the patient in a viral vector for gene therapy can be selected by a person skilled in the art. These therapeutic nucleic acid sequences typically encode products (eg, proteins or RNA) for administration and expression in a patient in vivo or ex vivo for the treatment of an inherited or non-inherited genetic defect, for example, by replacing or correcting a deficiency, for treating an epigenetic disorder or a disease or for treating a condition associated with dysregulation of a gene product. Such therapeutic genes that are desirable for gene therapy include, but are not limited to, the very low density lipoprotein receptor (VLDL-R) gene for the treatment of familial hypercholesterolemia or familial combined hyperlipidemia, the cystic fibrosis transmembrane regulator (CFTR) gene for the treatment of cystic fibrosis, allele Becker gene DMD for the treatment of Duchenne muscular dystrophy, and a number of other genes that can be easily selected by a person skilled in the art for the treatment of a specific disorder or disease. In a preferred embodiment, the rAAV vector contains a transgene encoding a therapeutic protein or RNA, such as miRNA. Preferably, the therapeutic protein is selected from the group consisting of factor IX (preferably human factor IX), factor VIII (preferably human factor VIII), lipoprotein lipase (LPL; including mutants such as, for example, LPL ^S447X ; see WO 01/00220 A2), porphobilinogen deaminase (PBGD), very low density lipoprotein receptor (VLDL-R), cystic fibrosis transmembrane conductance regulator (CFTR), product of the Becker allele of the Duchenne muscular dystrophy gene (DMD), product of the hyperoxaluria gene (AGXT), N-acetyl -alpha-D-glucosaminidase (NaGlu), glial cell line-derived neurotrophic factor (GDNF), S100A1 (also known as calcium binding protein A1 S100, which in humans is encoded by the S100A1 gene). In a preferred embodiment, the therapeutic protein is factor IX, more preferably human factor IX.

Alternatively or in combination with any of the preceding embodiments, in a preferred embodiment, gene therapy is intended to treat, prevent, cure and/or reverse a condition or disease, preferably a so-called orphan disease, which is understood herein as a rare disease affecting a small the percentage of a population, for example, less than 1 in 1,500 people in a population that is life-threatening, chronically debilitating, and/or inadequately treated. Typically, an orphan disease is a genetic disease and thus a lifelong disease, even if symptoms do not appear immediately. In a preferred embodiment, the condition or disease is selected from the group consisting of 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 congenital amaurosis, X-linked severe combined immunodeficiency (SCID), severe combined immunodeficiency with adenosine deaminase deficiency (ADA-SCID), adrenoleukodystrophy, chronic lymphocytic leukemia, acute lymphocytic leukemia, multiple myeloma, cystic fibrosis for , sickle cell disease, hyperlipoproteinemia type I, thalassemia, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), epilepsy, Friedreich's ataxia, Fanconi anemia, Batten disease, wet AMD, alpha-antitrypsin-1 deficiency, 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 (eg, Wilson's or Menkes disease), lysosomal acid lipase deficiency, hypooxaluria, Gaucher disease, Hurler disease, adenosine deaminase deficiency, glycogen storage disease and retinal degenerative disease (such as RPE65 deficiency, choroideremia).

In a further embodiment, said AAV5 gene therapy vector is for use in the human medical treatment of the invention, wherein said use includes administration into the bloodstream, eg, administration of the AAV5 gene therapy vector into the bloodstream. Blood may contain antibodies against AAV5, and in particular, a method of delivery through the bloodstream, for example, by intravascular infusion or injection, is provided. Delivery through the bloodstream allows delivery of the AAV5 vector to the target tissue. Such delivery to the target tissue can be achieved through systemic delivery. The present invention is not limited to administration into the bloodstream. Indeed, conventional and pharmaceutically acceptable routes of administration that may be contemplated include direct delivery to an organ, tissue or target site (eg, liver or CNS), intranasal, intravenous, intramuscular, subcutaneous, intradermal, oral, and other parenteral routes of administration. However, a preferred target tissue that may be provided is the liver. Thus, most preferably, the AAV5 gene therapy vector delivers its transgene to the liver via a bloodstream route. As stated, the AAV5 viral vector is administered in sufficient quantities to transfect the desired cells and provide sufficient levels of transduction and expression of the selected transgene to provide therapeutic benefit without undue adverse effects or with medically acceptable physiological effects as may be determined by one skilled in the art. in medecine. Methods of administration may also be combined if desired. Dosages of the rAAV vector (ie, the AAV5 gene therapy vector) are primarily dependent on factors such as the condition being treated, the gene selected, the age, weight, and health status of the patient, and may thus vary among patients.

The AAV5 gene therapy vector for use in human medical treatment of the invention is preferably an AAV5 gene therapy vector produced in insect cells. Without being bound by theory, the method of production may play a role in the immune profile associated with the AAV vector, since AAV capsids may differ when produced in insect cells from AAV capsids produced in mammalian cells. This difference may be present in relation to glycosylation or other post-translational modification. In addition, mammalian cell-based production may have the side effect that rep and cap expression constructs are contained within AAV capsids that are administered to patients, resulting in the transfer of rep and cap expression constructs, albeit in very small quantities, to human subjects. Expression of AAV rep and cap in a human patient may be immune detrimental, particularly in the case of human patients who test positive for anti-AAV5 antibodies. Thus, it may be preferable that the AAV5 viral vector intended for administration to human patients be produced in insect cells. Production from insect cells is well established and includes, but is not limited to, means and methods as described in WO 2007046703, WO 2007148971, WO 2009014445, WO 2009104964, WO 03042361, WO 2008024998, WO 2010114948, the contents of which is given in this description as links.

In another embodiment, the present invention provides a method for determining human patients suitable for medical treatment using an AAV5 gene therapy vector, comprising the steps of:

obtaining a serum sample from a human patient;

determination of antibody titer against AAV5;

wherein patients may be considered suitable for medical treatment if the anti-AAV5 total antibody titer is in the range of 0.02-5, as determined using the anti-AAV5 total antibody titer (TAb) assay as described in the Examples.

Optionally, said method then includes the step of:

administering the AAV5 gene therapy vector to a suitable human patient.

It is understood that any of the requirements and limitations as described above with respect to embodiments relating to the medical use of AAV5 gene therapy factors also apply to any of the methods as described herein, for example, methods for delivering an AAV5 gene therapy vector or for determining suitability . Preferably, said total anti-AAV5 antibody titer is in the range of 0.02-4, 0.02-3, or 0.02-2, as determined for total anti-AAV5 antibodies (TAbs) as described in the examples.

In another embodiment, a method for determining human patients suitable for medical treatment using an AAV5 gene therapy vector includes the steps of:

obtaining a serum sample from a human patient;

determination of antibody titer against AAV5;

wherein patients may be considered eligible for medical treatment if the anti-AAV5 neutralizing antibody titer is in the range of 3-10,000, as determined using an anti-AAV5 neutralizing antibody (NAb) assay as described in the Examples.

Optionally, said method then includes the step of:

administering the AAV5 gene therapy vector to a suitable human patient.

Preferably, said anti-AAV5 antibody titer is in the range of 3-5000, 33000, or 3-1000, as determined using an anti-AAV5 neutralizing antibody (NAb) assay as described in the examples.

It is understood that the above-described eligibility criterion is not the only criterion that can be used to select gene therapy treatments using AAV5. Thus, when a human patient satisfies all other criteria, the anti-AAV5 antibody criterion determines the eligibility of the human patient.

In another embodiment, the present invention provides a method of treating a human, comprising administering an effective amount of an AAV5 gene therapy vector to a human in need thereof;

wherein the individual is not pre-screened using an anti-AAV5 antibody assay;

and where said person has not been subjected to medical treatment using the AAV5 gene therapy vector prior to said medical treatment.

In a further embodiment, the present invention provides a method of treating a human, comprising administering an effective amount of an AAV5 gene therapy vector to a human in need thereof;

wherein said person is pre-screened using an anti-AAV5 antibody assay;

wherein said person has not been subjected to medical treatment using the AAV5 gene therapy vector prior to said medical treatment;

and wherein said individual has a level of anti-AAV5 antibody corresponding to at most the 95th percentile of anti-AAV5 antibody levels as observed in the human population.

In another further embodiment, the present invention provides a method of gene delivery to a human, comprising administering an effective amount of an AAV5 gene therapy vector to a human in need thereof;

wherein said person is pre-screened using an anti-AAV5 antibody assay;

wherein said person has not been subjected to medical treatment using the AAV5 gene therapy vector prior to said medical treatment;

and wherein said individual has an anti-AAV5 antibody level corresponding to at most the 95th percentile of anti-AAV5 antibody levels as observed in the human population.

In another embodiment, the present invention provides a method of gene delivery to a human, comprising administering an effective amount of an AAV5 gene therapy vector to a human in need thereof;

wherein the individual is not pre-screened using an anti-AAV5 antibody assay;

wherein said individual has not been subjected to medical treatment using the AAV5 gene therapy vector prior to said medical treatment.

Alternatively or in combination with any of the preceding embodiments, in a preferred embodiment, the AAV5 vector composition further comprises a pharmaceutically acceptable carrier, diluents, solubilizer, excipient, preservative and/or excipient. The rAAV vector carrying the therapeutic gene can be administered to a patient, preferably suspended in a biocompatible solution or pharmaceutically acceptable delivery vehicle. A suitable carrier includes sterile saline solution. Other aqueous and non-aqueous isotonic sterile injection solutions and aqueous and non-aqueous sterile suspensions known to be pharmaceutically acceptable carriers and well known to one skilled in the art can be used 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 to provide therapeutic benefit without undue adverse effects or with medically acceptable physiological effects as may be determined by one skilled in the art. in medecine.

Examples

Study design and participants.

A multinational, open-label, phase 1/2 dose-escalation study was conducted in adult men with severe (FIX <1 IU/dL) or moderate-severe (FIX <2 IU/dL) hemophilia B who required either: 1) continuous prophylaxis FIX, or 2) administration of FIX as needed and who have either >4 bleeds per year or hemophilic arthropathy. Additional details of the study can be found on the NIH clinicaltrials.gov website (NCT02396342). The study was approved by the institutional review board/institutional ethics committee at each center. All participants provided written informed consent. The study was conducted in accordance with the Declaration of Helsinki and the principles of Good Clinical Practice.

An AAV5 vector incorporating the codon-optimized wild-type hFIX gene under the control of the liver-specific LP1 promoter (Nathwani et al. N Engl J Med 2011; 365(25): 235765 and Nathwani et al. B. N 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 practice. The genomic copy titers (gc) of the vector were determined using qPCR. The ratio of capsid to gk was approximately 10, i.e. the number of capsids was approximately ten times the number of genomic copies. The 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 was chosen for its capacity to separate AAV particles from smaller matrix components. In this method, a calibration curve is obtained using an AAV vector preparation with a known total particle concentration. The total number of injected particles is plotted against the response data on the calibration curve. Using the obtained AAV peak area and the calibration curve, the number of injected sample particles is calculated by interpolation. The AAV5 vector is administered as a single, 30-minute, peripheral intravenous infusion. Participants were treated in two sequential dose escalation cohorts: Cohort 1

- 10 044191 (n=5, participants 1-5), in which 5x10 ¹² gc/kg was administered, and cohort 2 (n=5, participants 6-10) - 2x10 ¹³ gc/kg. Cohort 1 consisted of adult men with a mean age of 69 years (35-72) and a mean body weight of 84.5 kg (71.2-89.1), Cohort 2 consisted of adult men with a mean age of 35 years (33-46) and an average body weight of 84.0 kg (71.4-96.0). Efficacy outcome measures included plasma FIX activity measures. In addition, serum from subjects was obtained for analysis of anti-AAV5 neutralizing antibody titer (NAb titer) and total anti-AAV5 antibody titer (TAb).

Control serum from healthy donors was obtained commercially from SeraLab (West Sussex, UK). All information provided related to this serum is listed below.

Table 1

Healthy donors

Control serum THE CONSIGNMENT FLOOR: WHO ASD T: THESE,: THE CONSIGNMENT FLOOR: WHO ASD T: ETN: 1 MAN 33 NEGROID 26 MEN'S OH 57 EUROPIO id 2 MAN 53 NEGROID 27 MEN'S OH 59 EUROPIO id 3 MAN 41 NEGROID 28 MEN'S OH 54 EUROPIO id 4 MAN 49 HISPANIC 29 MAN oh 48 EUROPIO id 5 MAN 59 HISPANIC thirty MAN oh 41 EUROPIO id 6 MUSHS 45 HISPANIC 31 MEN'S 52 EUROPIO

- 11 044191

COY OH ID 7 MAN 41 HISPANIC 32 MAN oh 57 EUROPIO ID 8 MAN 23 HISPANIC 33 MAN oh 52 EUROPIO ID 9 MAN 39 NEGROID 34 MAN oh 50 EUROPIO ID 10 MAN 37 NEGROID 35 MAN oh 51 EUROPIO ID AND WOMEN II 38 NEGROID 36 WOMEN'S 24 EUROPIO ID 12 WOMEN II 27 HISPANIC 37 WOMEN'S 28 EUROPIO ID 13 WOMEN II 48 HISPANIC 38 WOMEN'S 54 EUROPIO ID 14 WOMEN II 49 EUROPIOI D 39 WOMEN'S 23 EUROPIO ID 15 WOMEN II 31 NEGROID 40 WOMEN'S 47 EUROPIO ID 16 WOMEN II 23 HISPANIC 41 WOMEN'S 27 EUROPIO ID 17 WOMEN II 45 NEGROID 42 FEMININE Y 39 EUROPIO ID 18 WOMEN II thirty HISPANIC 43 FEMININE Y 35 EUROPIO ID 19 WOMEN II 36 NEGROID 44 FEMININE Y 24 EUROPIO ID 20 WOMEN II 25 HISPANIC 45 FEMININE Y 47 EUROPIO ID 21 MUSHS COY 59 EUROPIOI D 46 FEMININE Y 57 EUROPIO ID 22 MAN 50 EUROPIOI D 47 FEMININE Y 55 EUROPIO ID 23 MUSHS 54 EUROPIOI 48 FEMININE 46 EUROPIO whoa D Y ID 24 MAN 57 EUROPIOI D 49 FEMININE Y 21 EUROPIO ID 25 MAN 65 EUROPIOI D 50 FEMININE Y 51 EUROPIO ID

Anti-AAV5 neutralizing antibody (NAb) titer.

Nab levels in human serum were assessed based on a highly sensitive in vitro assay using AAV5 carrying a luciferase transgene (AAV5-luc) and the human embryonic kidney cell line HEK293T (ATCC 11.268). Transgene expression was detected by addition of a luciferin analogue.

- 12 044191

Used materials:

HEK293T cells (HEK293T/ATCC 11.268);

DMEM with phenol red (Gibco, REF# 31966)/10% FBS (Greiner, REF# 758093)/1% pen-strept.

(Gibco, REF# 15140);

DMEM without phenol red (Gibco, REF# 21063)/l% pen.-strept. (Gibco, REF# 15140);

1xPBS-/- (Gibco, REF# 14190);

1x trypsin-EDTA (Gibco, REF# 25200);

solution (2.5%) poly-L-lysine (PLL) (Sigma-Aldrich, REF# 8920-100);

96-well flat-bottomed black culture plates (costar, REF #3916);

clear 96-well flat bottom plates (corning, REF #3596);

ONE-Glo Luciferase Assay System (Promega, REF# E6120);

Glo lysis buffer, 1x (Promega, REF# E2661);

used AAV5-CMV-luc (eg, AAV5-CMV-73QlucHtt from PKO, titer: 4e13 gc/ml).

Basically, cells were seeded into black 96-well plates and clear 96-well plates by adding 100 μl/well of HEK293T cells in DMEM (with phenol red/10% FBS and 1% P/S (penicillin/streptomycin)) at a concentration of 0 .5x105 cells/well. Cells were incubated overnight.

The next day, serial dilutions of plasma were obtained in transparent 96-well overnight plates in medium (DMEM/1% PS without phenol red/10% FBS). The resulting final plasma dilutions, after addition of the virus (see below), were 2, 4, 8, 16, 32, 64, 128, 256, 512 and 1024.

Dilutions were made by adding 140 µl of medium to the wells designated A2-A11 (negative control wells), and 70 µl of medium was added to the remaining rows in columns 3, 4, 5, 6, 7, 8, 9, 10 and 11 of the plate, and in the row H2-H11 (positive control). Samples are added to wells B2, C2, D2, E2, F2, G2 (140 µl/well), resulting in the first dilution: 1. Serial serial dilutions of plasma are performed in the plate by transferring 70 µl from column 2 to column 3 (dilution 2 ), from 3 to 4 (4), from 4 to 5 (8), from 5 to 6 (16), from 6 to 7 (32), from 7 to 8 (64), from 8 to 9 (128), from 9 to 10 (256), from 10 to 11 (512), then 70 µl from column 11 is discarded. AAV5-CMV-73QlucHtt is prepared in medium (DMEM/1% PS without phenol red/10% FBS) at 6x109 gq/ml. Then 70 μl/well of the virus dilution 6x109 gc/ml AAV5(160)-CMV-73QlucHtt is added to the plasma dilution plates, excluding wells A2-A11 (negative control). The plates are carefully placed in a plate shaker for 2 min at 300 rpm. The plates are then incubated for 1 hour at 4°C.

The culture medium is removed from the black 96-well plates prepared the previous day (with HEK293T cells) and the prepared plasma dilutions are transferred by pipetting from transparent plates into black plates with HEK293T cells in a volume of 100 μl/well. These plates were incubated for 16-20 hours at 37°C. The next day, the cells were equilibrated at room temperature and the medium was removed. Cells were washed once with 1X PBS−/− (100 μl/well), after which 100 μl/well Glo lysis buffer was added to the plates and incubated for 5 minutes at room temperature to allow lysis to occur. Next, add 100 µl/well of reagent from the ONE-Glo Luciferase Assay System (reagent prepared according to manufacturer's instructions). After at least 3 minutes, measure the plates using the ONE-Glo protocol on the GloMax Discover device. Anti-AAV5 neutralizing antibody titers are determined using a LabKey software assay that calculates the percent neutralization for each serum dilution after subtracting background activity and then fits a curve to the neutralization profile. It then uses this curve to calculate neutralizing antibody titers for the selected reference, area under the curve (AUC), and error estimates. The four-parameter method was used to calculate curve fitting. LabKey calculates the IC50, the dilution at which the antibody inhibits transduction by 50%. LabKey also calculates point titers, according to Johnson and Byington, Techniques in HIV Research. New York, N.Y.: Stockton Press, 1990: 71-76. This is done by linear interpolation between two replicates on either side of the target percent neutralization. Each assay included a positive control (wells with no serum sample but with AAV5-LUC), a negative control (wells containing media only, no serum sample, and no AAV5-LUC), and a negative serum control (heat-inactivated FBS) to assess neutralization specificity AAV5-LUC. FBS should not have anti-AAV5 neutralizing properties when measured as a sample.

Antibody titer against AAV5.

Quantification of total human anti-AAV5 Abs was based on an ELISA assay using a specific plate-coating capsid. The presence of total human Abs specific against the AAV5 capsid was detected using protein A peroxidase. ELISA plates (Nunc MaxiSorp plate. Ref: 456537, Thermo Scientific) were coated with antigen (cap AAV5) at 100 ng/well in carbonate buffer overnight at 4°C. The next day, the plates were washed three times with PBS tween-20 (PBSt) to remove residual antigen and blocked using blocking solution (PBS+3% FBS) to prevent nonspecific binding. After washing three times with 200 μl of PBSt, dilutions of human serum in PBSt were added starting at 1:9, followed by serial dilutions of 1:3 in a final volume of 100 μl. All samples were tested in duplicate. A negative control without human serum was included in each plate. Serum dilutions were incubated for 2 hours at 37°C. After this, the serum was removed, the plate was washed three times with PBSt, and 100 μl of protein A with peroxidase diluted 1:10,000 in blocking solution was added for one hour. The plate was washed three times using PBSt and the reaction was detected using TMB substrate and stopped after 30 min using 2H H ₂ SO ₄ . The absorbance was read at 450 nm in a microplate reader. The total antibody titer was calculated as a serum dilution having an optical density five times greater than the negative control.

Results and discussion

All human patients in both cohorts showed significant improvements in FIX activity, with most human patients improving through a change in phenotype from severe to mild (Table 2), resulting in a significant reduction or even elimination of the use of prophylactic FIX protein administration. There was observed variability between FIX activity levels between patients and between cohorts. Variability in FIX activity levels did not correlate with NAb or TAb status of human patients.

The previously published prevalence of anti-AAV5 TAbs (40%, Boutin et al. Hum Gene Ther. 2010 Jun 21(6): 704-712) was approximately consistent with the results of the present analysis (30%). Results obtained using the luciferase-based Nab assay (see Figures 1 and 2) suggest that the prevalence of (neutralizing) anti-AAV5 antibodies is similar, as 14 of the 50 control serum samples screened had a positive signal (28% ), which is consistent with recent studies (Li C et al. Gene Ther. 2012 Mar;19(3):288-94). The results for serum obtained from human patients prior to gene therapy treatment are also consistent with this, where 3 out of 10 serum samples were found to be positive in both the Nab and Tab assays (30%). Total antibodies, as assessed by ELISA, and neutralizing antibodies, as assessed by luciferase-based assay, correlated closely, suggesting that both assays detect the same molecule (see Fig. 2A).

In addition, the presence of neutralizing antibody titers after treatment was also tested and found to be in the range of approximately 106 and above. Thus, the antibody titers found in untreated, endemically acquired individuals are well outside the range of titers observed in human patients treated with AAV5-based gene therapy. In addition, patients with pre-existing anti-AAV5 NAbs show a rapid increase in IgG levels after administration of AAV-FIX, characteristic of an immune booster, in contrast to patients without NAbs who show a rapid and transient increase in IgM levels, followed by an increase in IgG levels , which is typical for the first exposure to an antigen. In addition, there was no evidence of increased ALT (alanine aminotransferase) levels or activation of capsid-specific T cells in treated patients with preexisting NAbs. In summary, exposure to AAV5-based gene therapy in patients with endemically acquired preexisting NAbs was well tolerated without increasing ALT levels or T-cell activation.

In conclusion, the presence of anti-AAV5 antibodies detected in vitro by either the Nab assay or the TAB assay was neither predictive nor indicative of impaired transduction in vivo. There was no evidence of a correlation between the presence of Nab before therapy and FIX levels after therapy, as a result of FIX gene transfer by AAV5. Strikingly, the individual with the highest response in cohort 1, who received a lower dose of AAV5 vector, also had the highest detectable levels of NAb and TAb antibodies. The range of anti-AAV5 antibody titers observed in the healthy population indicates that antibody levels in the healthy population that have not been subjected to AAV5 gene therapy do not impair AAV5 transduction in vivo. This occurs because the highest titer observed in the healthy population lies close within the range of the highest titer observed in patient 5 of cohort 1. Thus, it is believed that testing for the presence or absence of anti-AAV5 antibodies in an untreated population is not necessary before treatment using the AAV5 gene therapy vector.

Table 2. Average levels of FIX in the equilibrium state and preventive status of participants. Only values at least 10 days after the last FIX administration were included; ^a The prophylactic status is the same as at the last examination. CI, confidence interval; FIX, factor nine; ME, international units.

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Claims (11)

table 2 Participant Before treatment After treatment Activity FIX, IU/dl Phenotype hemophilia B Prophylactic administration of FIX Average FIX level at equilibrium, activity, IU/dL (95% CI) Prophylactic administration FIX' Phenotype hemophilia B Reducing severity 1 <1 Heavy Yes 6.2 (5.8-6.6) No Soft Yes 2 <1 Heavy Yes 4.7 (4.5-5.0) No Moderate Yes 3 <1 Heavy Yes 1.3 (-0.7-3.2) Yes Moderateheavy Yes 4 1.5 Moderateheavy Yes 6.8 (6.3-7.3) No Soft Yes 5 <1 Heavy Yes 3.0 (2.6-3.4) No Moderate Yes 6 <1 Heavy Yes 12.7(11.9-13.5) No Soft Yes 7 Heavy Yes 6.4 (6.0-6.7) No Soft Yes 8 <1 Severe ^No 6.8 (5.8-7.7) No Mild Yes 9 <1 Heavy Yes 3.1 (2.8-3.3) No Moderate Yes 10 <1 Heavy Yes 5.8 (5.4-6.2) No Soft Yes CLAIM 1. The use of an AAV5 gene therapy vector in the medical treatment of a person suffering from a disease that can be ameliorated by AAV-based gene therapy, wherein the person is not pre-screened using an anti-AAV5 antibody assay and where the person has not been subjected to medical treatment using a gene therapy AAV5 vector prior to indicated medical treatment. 2. The use of an AAV5 gene therapy vector in the medical treatment of a person suffering from a disease that can be ameliorated by AAV-based gene therapy, wherein the person has been prescreened using an anti-AAV5 antibody assay and where the person has not been medically treated using the gene therapy vector AAV5 prior to specified medical treatment, wherein the individual has an anti-AAV5 antibody level corresponding to at most the 95th percentile of anti-AAV5 antibody levels as observed in a human population, and wherein the individual is tested positive for anti-AAV5 antibodies. 3. Use according to claim 1 or 2, wherein said AAV5 gene therapy vector is administered at a dose corresponding to at least 10 ¹² capsids/kg. 4. Use according to any one of claims 1 to 3, wherein said AAV5 gene therapy vector is used at a dose corresponding to at least 10 ¹² gc/kg body weight. 5. Use according to any one of claims 1 to 4, wherein said AAV5 gene therapy vector is used in the treatment of a disease selected from the group consisting of hemophilia A or hemophilia B. 6. Use according to any one of claims 1 to 5, wherein said AAV5 gene therapy vector is used in the treatment of hemophilia, wherein the AAV5 gene therapy vector encodes a FIX protein or a variant thereof. 7. Use according to any one of claims 1 to 6, wherein said use includes introducing the vector into the bloodstream. 8. Use according to any one of claims 1 to 7, wherein said use includes delivery of the vector to the liver. 9. Use according to any one of claims 1 to 8, wherein said AAV5 gene therapy vector is produced in insect cells. 10. A method for identifying human patients suitable for medical treatment using an AAV5 gene therapy vector, comprising the steps of: obtaining a serum sample from a human patient; determination of antibody titer against AAV5; wherein patients may be considered suitable for medical treatment if the anti-AAV5 total antibody titer is in the range of 0.02-5, as determined using an anti-AAV5 total antibody (TAb) assay. 11. A method for identifying human patients suitable for medical treatment using an AAV5 gene therapy vector, comprising the steps of: obtaining a serum sample from a human patient; determination of antibody titer against AAV5; wherein patients can be considered suitable for medical treatment if the anti-AAV5 antibody titer is in the range of 3-5000, as determined using an anti-AAV5 neutralizing antibody assay, as determined by a Nab assay. -