Co-treatment for gene therapy

Abstract

The present invention relates to a method for enhancing gene therapy by using effective enzymes to degrade or digest serum antibodies and / or inhibit or reduce the effector function of serum antibodies in a subject. More specifically, the subject either has no detectable antibodies that specifically bind to the gene therapy agent, or has a low titer of neutralizing antibodies (NAbs) that specifically bind to the gene therapy agent, or a low titer of total antibodies (TAbs) that specifically bind to the gene therapy agent. The method extends the serum half-life of the therapeutic agent, meaning that the gene therapy agent can be administered at a lower dose than would otherwise be administered.

Description

[Technical Field]

[0001] The present invention relates to gene therapy and includes compositions and methods for improving gene therapy, particularly in terms of reducing the dose of gene therapy agent required and / or prolonging the effectiveness of gene therapy. [Background technology]

[0002] Gene therapy is a rapidly developing and very promising approach, for example, for the treatment of monogenic diseases. However, one of the challenges facing gene therapy is that the body often elicits an immune response to the gene therapy agent, especially when repeated doses of the gene therapy agent are administered. Some gene therapy vectors are based on naturally occurring viruses such as adeno-associated viruses (AAV), and infection with wild-type AAV is common, which is why subjects may already have antibodies specific to the gene therapy agent before gene therapy administration.

[0003] Antibodies against therapeutic drugs, especially against gene therapy agents, are known as anti-drug antibodies (ADA). The production of ADA can not only reduce the transduction efficiency of gene therapy but also promote the rapid clearance of gene therapy agents. Therefore, ADA can reduce the efficiency of gene therapy agents. Some individuals have such high pre-existing ADA titers that administering gene therapy agents as treatment is considered completely inappropriate for them. For those individuals whose ADA titers become so high after the first gene therapy treatment, administering additional doses of gene therapy is no longer considered safe, meaning that the treatment, which was initially effective, is no longer appropriate.

[0004] ADAs, whether pre-existing or increasing after an initial gene therapy treatment, can interfere with the effectiveness of gene therapy in certain individuals. Furthermore, their presence can necessitate the administration of high titers of gene therapy vectors to achieve the desired effect, which can pose safety concerns. Given the history of dose-related adverse events in gene therapy, safety is a key factor, and anything that allows for a lower dose of administered gene therapy agent is clearly desirable.

[0005] Therefore, taking into account the effects of ADA, attempts to find ways to reduce the effects of ADA in gene therapy have been a continuing focus. Summary of the Invention [Problem to be solved by the invention]

[0006] Surprisingly, it has been found that even in subjects with low or no serum antibodies to gene therapy agents, administration of an enzyme capable of targeting and degrading serum antibodies prior to administration of the gene therapy agent is even more beneficial. Considering that ADA is generally believed to be the cause of reduced efficiency of gene therapy, the discovery that there may be benefits to administering an enzyme that targets serum antibodies in addition to the gene therapy agent, even in the absence or low levels of antibodies specific to the gene therapy agent, is surprising in light of the attention to ADA in the field.

[0007] It has also been unexpectedly discovered that this enzyme may slow the clearance of gene therapy, particularly from the circulation, in subjects generally, thereby increasing the subject's exposure to the gene therapy agent. Thus, the present invention can also provide a method for reducing the dose of a gene therapy agent to be administered to a subject by increasing the subject's exposure to the gene therapy agent.

[0008] Thus, the present invention provides a method for enhancing gene therapy in general, and also for facilitating treatment for specific patients who were previously thought to avoid the need for administration of the enzyme during gene therapy. The cleavage products of the enzyme appear to have an unexpected inhibitory effect on the clearance of gene therapy vectors. By delaying clearance of gene therapy vectors and maintaining the vector in the circulation, delivery to target organs can be improved.

[0009] Therefore, the advantages of the present invention include the ability to reduce the amount of gene therapy agent administered, for example, to a dose lower than that normally administered. Another additional advantage may be the ability to extend the half-life of the administered gene therapy agent in the body. Another advantage may be the ability to obtain a greater effect for an administered amount of gene therapy agent. Another advantage may be the ability to increase the subject's exposure to gene therapy. Another advantage may be the ability to delay the clearance time of the gene therapy agent. Another advantage may be the ability to increase the amount of gene therapy agent reaching the target organ. Another advantage may be the ability to treat patient groups for which administration of such enzymes was previously considered unnecessary. Another advantage may be the ability to increase the rate of gene transfer. These advantages may occur alone or in combination with each other.

[0010] Thus, the present invention provides a method of treating a subject in need of gene therapy for a disease, comprising the steps of: a. administering to the subject an enzyme in an amount effective to degrade or digest serum antibodies and / or inhibit or reduce effector functions of serum antibodies in the subject; and b. administering to the subject a gene therapy agent; wherein the gene therapy agent is administered at a dose that is less than the minimum effective dose (MED) that would be administered to the subject in the absence of step a; The subject has either no detectable antibodies that specifically bind to the gene therapy agent, or a low titer of neutralizing antibodies (NAb) that specifically bind to the gene therapy agent, or a low titer of total antibodies (TAb) that specifically bind to the gene therapy agent, and optionally, the presence / titer of antibodies in the subject is measured in a serum sample obtained from the subject prior to step a.

[0011] The present invention further provides a gene therapy agent for use in a method of treating a subject in need of gene therapy for a disease, comprising the steps of: a. administering to the subject an enzyme in an amount effective to degrade or digest serum antibodies and / or inhibit or reduce effector functions of serum antibodies in the subject; and b. administering to the subject a gene therapy agent; wherein the gene therapy is administered at a dose that is less than the minimum effective dose (MED) that would be administered to the subject in the absence of step a; The subject has either no detectable antibodies that specifically bind to the gene therapy agent, or a low titer of neutralizing antibodies (NAb) that specifically bind to the gene therapy agent, or a low titer of total antibodies (TAb) that specifically bind to the gene therapy agent, and optionally, the presence / titer of antibodies in the subject is measured in a serum sample obtained from the subject prior to step a.

[0012] The present invention further provides an enzyme for use in a method of treating a subject in need of gene therapy for a disease, comprising the steps of: a. administering to a subject an amount of an enzyme, wherein the enzyme is effective to degrade or digest serum antibodies and / or inhibit or reduce effector functions of serum antibodies in the subject; and b. administering to the subject a gene therapy agent; wherein the gene therapy is administered at a dose that is less than the minimum effective dose (MED) that would be administered to the subject in the absence of step a; The subject has either no detectable antibodies that specifically bind to the gene therapy agent, or a low titer of neutralizing antibodies (NAb) that specifically bind to the gene therapy agent, or a low titer of total antibodies (TAb) that specifically bind to the gene therapy agent, and optionally, the presence / titer of antibodies in the subject is measured in a serum sample obtained from the subject prior to step a.

[0013] The reaction products resulting from the activity of the enzyme may increase the serum half-life of the gene therapy agent in the subject and / or may increase or prolong the subject's exposure to the gene therapy agent. [Brief explanation of the drawings]

[0014] [Figure 1] qPCR detection of AAV in SCID mouse plasma up to 7 days after AAV injection. SCID mice were administered either: (a) AAV gene therapy alone (circles); (b) AAV gene therapy with human IVIg (squares); or (c) IdeS and IVIg with AAV gene therapy. AAV levels in mouse serum were measured by qPCR up to 7 days after AAV gene therapy administration and the results are shown in Figure 1. [Figure 2] qPCR detection of AAV in SCID mouse plasma up to 48 hours after AAV injection. Results from the same experiment as in Figure 1, but showing serum AAV levels up to 48 hours. [Figure 3] qPCR detection of AAV in SCID mouse plasma at 1, 4, 24, and 48 hours after AAV injection. Results of the same experiment as in Figures 1 and 2, but with serum AAV levels at each time point shown as a bar graph. [Figure 4] qPCR detection of AAV in SCID liver cells 14 days after AAV injection. Results of the same experiment as in Figures 1-3, but showing AAV levels in liver cells measured 14 days after AAV administration. [Figure 5]qPCR detection of AAV in SCID cardiac cells 14 days after AAV injection. Results of the same experiment as in Figures 1-4, but showing AAV levels in cardiac cells measured 14 days after AAV administration.

[0015] A brief description of the sequence SEQ ID NO: 1 is the complete sequence of IdeS, including the N-terminal methionine and signal sequence. It is also available as NCBI reference sequence number WP_010922160.1. SEQ ID NO: 2 is the mature sequence of IdeS lacking the N-terminal methionine and signal sequence. It is also available as Genbank accession number ADF13949.1. SEQ ID NO: 3 is the complete sequence of IdeZ, including the N-terminal methionine and signal sequence. It is also available as NCBI reference sequence number WP_014622780.1. SEQ ID NO: 4 is the mature sequence of IdeZ lacking the N-terminal methionine and signal sequence. SEQ ID NO: 5 is the sequence of the IdeS / Z hybrid. The N-terminus is based on IdeZ, lacking the N-terminal methionine and signal sequence. SEQ ID NOs: 6-25 are exemplary protease sequences for use in the methods of the present invention. SEQ ID NO: 26 is the IdeS polypeptide sequence, which comprises the sequence of SEQ ID NO: 2 with an N-terminal methionine and histidine tag added (internal reference pCART124). SEQ ID NO: 27 is the sequence of the IdeZ polypeptide, which comprises the sequence of SEQ ID NO: 4 with an N-terminal methionine and histidine tag added (internal reference pCART144). SEQ ID NO: 28 is the sequence of the IdeS / Z polypeptide, which comprises the sequence of SEQ ID NO: 5 with an N-terminal methionine and histidine tag added (internal reference pCART145). SEQ ID NO:29 is the consecutive sequence PLTPEQFRYNN, which corresponds to positions 63 to 73 of SEQ ID NO:3. SEQ ID NO:30 is the consecutive sequence PPANFTQG, which corresponds to positions 58 to 65 of SEQ ID NO:1. SEQ ID NO:31 is the consecutive sequence DDYQRNATEAYAKEVPHQIT, which corresponds to positions 35 to 54 of SEQ ID NO:3. SEQ ID NO:32 is the consecutive sequence DSFSANQEIRYSEVTPYHVT, which corresponds to positions 30 to 49 of SEQ ID NO:1. SEQ ID NOs: 33 to 55 are nucleotide sequences encoding the above-mentioned proteases. SEQ ID NOs: 56-69 are exemplary protease sequences for use in the methods of the present invention. SEQ ID NO:70 is the consecutive sequence NQTN, which corresponds to positions 336 to 339 of SEQ ID NO:1. SEQ ID NO: 71 is the consecutive sequence DSFSANQEIRYSEVTPYHVT, which corresponds to positions 30 to 49 of SEQ ID NO: 1. SEQ ID NOs: 72 to 86 are nucleotide sequences encoding the polypeptides disclosed herein. SEQ ID NO:87 is the sequence SFSANQEIRYSEVTPYHVT, which corresponds to positions 31 to 49 of SEQ ID NO:1. SEQ ID NO: 88 is the sequence DYQRNATEAYAKEVPHQIT and corresponds to positions 36 to 54 of the IdeZ polypeptide, NCBI reference sequence number WP_014622780.1. SEQ ID NO: 89 is the consecutive sequence DDYQRNATEAYAKEVPHQIT, which may be present at the N-terminus of a polypeptide of the invention. SEQ ID NO: 90 is the mature sequence of EndoS (Streptococcus pyogenes endoglycosidase). SEQ ID NO:91 and SEQ ID NO:92 are other exemplary proteases for use in the methods of the invention. SEQ ID NO:92 is similar to SEQ ID NO:91 except that it lacks the first 20 residues at the N-terminus of SEQ ID NO:91, which consists of the consecutive sequence DDYQRNATEAYAKEVPHQIT. SEQ ID NO: 93 is the amino acid sequence of the Spk1 protein. SEQ ID NO: 94 is the amino acid sequence of the Spk2 protein.

[0016] general It is to be understood that the various applications of the products and methods disclosed herein can be tailored to the particular needs of the art, and it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention and is not intended to be limiting.

[0017] Furthermore, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes "polypeptides," and the like.

[0018] The term "polypeptide" is used herein in its broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. Thus, the term "polypeptide" includes not only short peptide sequences but also longer polypeptides and proteins. As used herein, the term "amino acid" refers to any natural and / or unnatural or synthetic amino acid, including both D- and L-enantiomers, and also amino acid analogs and peptidomimetics.

[0019] The terms "patient" and "subject" are used interchangeably and typically refer to a human. References to IgG typically refer to human IgG unless otherwise specified.

[0020] Where the term "comprising" is used herein, the present invention also provides embodiments that "consist essentially of" and "consist of" what is set forth.

[0021] Amino acid identity as described above can be calculated using any suitable algorithm. For example, the PILEUP or BLAST algorithms can be used to calculate identity or align sequences to identify equivalent or homologous sequences (typical default settings), as described, for example, in Altschul SF (1993) J Mol Evol 36:290-300; Altschul SF et al. (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves initially identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in a query sequence that either match or satisfy some positive threshold score T when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds for initiating searches to find HSPs containing them. Word hits are extended in both directions along each sequence for as long as the cumulative alignment score increases. Extension for word hits in each direction is terminated when: the cumulative alignment score falls by an amount X from its maximum achieved value; the cumulative score becomes 0 or lower due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919), alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0022] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences; see, e.g., Karlin and Altschul (1993), Proc. Natl. Acad. Sci. USA, 90:5873-5787. One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two polynucleotide or amino acid sequences could occur by chance. For example, a sequence is considered to be similar to another sequence if the minimum sum probability in a comparison of a first sequence with a second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001. Alternatively, the UWGCG package provides the BESTFIT program (used, for example, by default) that can be used to calculate identity (Devereux et al. (1984), Nucleic Acids Research, 12, 387-395).

[0023] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0024] Enzyme treatment to enhance gene therapy The present invention provides methods for enhancing the efficiency of gene therapy. For example, the half-life of gene therapy may be extended by using an enzyme that can degrade or digest serum antibodies in a subject and / or inhibit or reduce the effector function of serum antibodies. By using such an enzyme, the dose of the gene therapy agent administered may be lower than the dose that would normally be administered without the enzyme. In some cases, the use of such an enzyme may enhance gene therapy in a subject even if the subject has low levels or no such antibodies. The subject's exposure to the gene therapy may be increased and / or extended.

[0025] Thus, the present invention provides a method of treating a subject in need of gene therapy for a disease, comprising the steps of: a. administering to a subject an amount of an enzyme effective to degrade or digest serum antibodies and / or inhibit or reduce effector functions of serum antibodies in the subject; and b. administering to said subject a gene therapy agent; wherein the gene therapy agent is administered at a dose that is less than the minimum effective dose (MED) that would be administered to the subject in the absence of step a; The subject has either no detectable antibodies that specifically bind to the gene therapy agent, or a low titer of neutralizing antibodies (NAb) that specifically bind to the gene therapy agent, or a low titer of total antibodies (TAb) that specifically bind to the gene therapy agent, and optionally, the presence / titer of antibodies in the subject is measured in a serum sample obtained from the subject prior to step a.

[0026] The reaction products resulting from the activity of the enzymes extend the serum half-life of the gene therapy agent in the subject and / or increase or extend the subject's exposure to the gene therapy agent.

[0027] Steps a and b may each be performed only once in the implementation of the method. Alternatively, steps a and b may be performed more than once, for example, two or more times. In some cases, they may be performed three, four, five or more times. The above-described method may be performed each time a subject requires gene therapy.

[0028] For example, steps a and b are typically performed in the order described, i.e., step a is performed first, followed by step b. In one example, steps a and b may be performed at intervals of several hours to one month. They may be performed at intervals of several hours to two weeks. They may be performed at intervals of four hours to one week. For example, they may be performed at intervals of four hours to 48 hours. In a preferred example, they may be performed at intervals of four hours to 24 hours. In a more preferred example, they may be performed at intervals of four hours to 12 hours. On the other hand, in some examples, the two steps may be performed simultaneously. In one embodiment, the two steps are performed simultaneously, separately, or sequentially. Typically, the two steps are provided as separate compositions. However, in some embodiments, the enzyme and the gene therapy agent are provided as the same composition. In yet another embodiment, the enzyme may be administered after gene therapy, for example, after the administration of the gene therapy agent within any of the time intervals described above.

[0029] In one embodiment, a measurement may be performed after step a to determine when to perform step b, e.g., the level of an antibody specific to the gene therapy agent may be measured. In one example, NAb may be measured. In another example, TAb may be measured. In one example, both may be measured. In another aspect, such a measurement may be performed before step a to determine whether the subject has no detectable antibodies that specifically bind to the gene therapy agent or has a low titer of NAb or a low titer of TAb; optionally, the presence / titer of antibodies in the subject may be measured in a serum sample obtained from the subject before step a.

[0030] In some instances where measurements are performed, the measurements are performed using a biological sample obtained from the subject. A preferred biological sample is blood or a blood product. In some embodiments, the blood product comprises plasma or serum. A particularly preferred sample is serum.

[0031] The present invention also provides methods for enhancing the delivery efficiency of gene therapy treatments.

[0032] The present invention also provides a method for treating a subject who does not exhibit a pre-existing ADA specific for a gene therapy agent or who has only a low level of ADA, comprising the steps of: a. administering to a subject an effective amount of an enzyme described herein; and b. Administering a gene therapy agent to the subject.

[0033] The present invention further provides a method for improving the efficiency of gene therapy, comprising the steps of: a. administering to a subject an effective amount of an enzyme described herein; and b. administering to the subject a gene therapy agent; A method wherein at least part of the enhanced efficacy is due to an effect separate from the treatment's effect on any ADAs present.

[0034] In one embodiment, the present invention is applicable to subjects who would not normally exhibit an ADA response and would therefore be administered only a gene therapy agent, but not an enzyme.

[0035] The present invention is applicable to any of the patient groups described herein. It can also be used as a method for enhancing gene therapy agents in patients in general. For example, the enzymes described herein can be used to prolong the clearance time of gene therapy agents. The enzymes described herein can be used to increase a patient's exposure time to a gene therapy agent. They can also be used to increase a patient's overall exposure to the gene therapy agent, i.e., in terms of exposure level and / or duration of exposure. They can also be used to increase the amount of gene therapy agent that reaches a specific target organ. For example, the present invention can be used to increase the amount of gene therapy agent that reaches the liver. It can be used to increase the amount of gene therapy agent that reaches the heart. It can be used to increase the amount of gene therapy agent that reaches muscle. It can also be used to extend the time that the gene therapy agent remains in these organs. Furthermore, the present invention can be used to prolong the time that the gene therapy agent remains in serum. It can be used to increase the level of the gene therapy agent present in serum. In one embodiment, increased levels of the gene therapy agent can be observed at least 48 hours after administration, for example, at least 24 hours after administration.

[0036] It should be understood that the increase or prolongation referred to herein is relative to a control method in which no enzyme is administered. The increase may be evaluated relative to a single control or relative to the average value of multiple experiments.

[0037] The present invention also provides methods for slowing the clearance of a gene therapy agent, including the following: a. administering to a subject an enzyme in an amount effective to degrade or digest serum antibodies in the subject and / or inhibit or reduce the effector function of serum antibodies; and b. Administering a gene therapy agent to said subject.

[0038] The gene therapy agent may be, for example, any of those described herein. The enzyme may also be, for example, any of those described herein. In one aspect, the clearance rate may be slowed overall for at least the first 0-72 hours compared to conditions in which the enzyme is not administered. In one aspect, the clearance rate may be slowed for at least 0-48 hours after administration of the gene therapy agent. In one aspect, the clearance rate may be slowed for at least 0-24 hours after administration of the gene therapy agent. In another aspect, the subject's overall exposure to the gene therapy agent may be increased for at least some period after administration of the gene therapy agent compared to conditions in which the enzyme is not administered. In some aspects, the timing of any of the administrations of the enzyme and / or gene therapy agent may be the timing of any of the other embodiments described herein.

[0039] In another aspect, the invention provides the use of an antibody cleavage product generated by any of the enzymes described herein to slow the rate of clearance of a gene therapy agent.

[0040] In a particularly preferred embodiment, the clearance rate or exposure is in the serum of a subject.

[0041] enzyme The enzymes utilized are typically capable of degrading or digesting serum antibodies in the subject and / or inhibiting or reducing the effector functions of serum antibodies. The term "serum antibodies in a subject" can refer to any gamma immunoglobulin (IgG1, IgG2, IgG3, and IgG4) molecules present in human tissues or in circulation before the method of the invention is performed.

[0042] The ability to degrade or digest serum antibodies and / or inhibit or reduce the effector functions of serum antibodies may refer to a reduction in the interaction of IgG molecules with Fc receptors. Here, the term "Fc receptor" refers to Fc gamma immunoglobulin receptors, i.e., FcγRs (Fc gamma receptors) present on cells. In humans, FcγR refers to one, some, or all of a family of receptors including FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIC (CD32C), FcγRIIIA (CD16a), and FcγRIIIB (CD16b). As used herein, the term FcγR also includes naturally occurring polymorphisms of FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIC (CD32C), FcγRIIIA (CD16a), and FcγRIIIB (CD16b).

[0043] The enzyme used in the methods of the present invention can be any enzyme that inactivates serum IgG, but is typically an IgG cysteine ​​protease that cleaves IgG so that the antibody-binding domain and the Fc-interacting domain are separated from each other. In some cases, the interaction of IgG molecules in serum with Fc receptors is reduced due to a decrease in the amount of unprocessed IgG molecules present in serum. As another example, the enzyme may be an IgG endoglycosidase that cleaves the glycan structure on the Fc-interacting domain of IgG, particularly the N-linked biantennary glycan present at position Asn297 (Kabat numbering). This glycan structure plays an important role in Fc receptor binding and complement activation. Therefore, if this glycan is completely or partially removed by a protein, even if the IgG molecule is intact, FcRn binding or complement activation is reduced, and the reduced FcRn binding also reduces recycling / half-life. Enzymes suitable for the present invention are described in more detail in the following sections.

[0044] The enzyme is preferably administered by intravenous infusion. The enzyme may be administered by any suitable route, including, for example, intradermal, subcutaneous, transdermal, intramuscular, intraarterial, intraperitoneal, intraarticular, intraosseous, intranasal, or other suitable route. The enzyme may be administered multiple times, for example, two, three, four, or more than three times. Preferably, the enzyme is administered twice.

[0045] The amount of enzyme that can be administered may be 0.01 mg / kg to 5 mg / kg body weight, 0.01 mg / kg to 4 mg / kg body weight, 0.01 mg / kg to 3 mg / kg body weight, 0.01 mg / kg to 2 mg / kg body weight, 0.01 mg / kg to 1 mg / kg body weight, 0.01 mg / kg to 0.5 mg / kg body weight, or 0.01 to 0.3 mg / kg body weight, preferably 0.1 to 0.3 mg / kg body weight, and more preferably 0.2 to 0.3 mg / kg body weight. In one example, the enzyme is administered in the range of 0.25 mg / kg to 0.5 mg / kg body weight, preferably 0.3 mg / kg to 0.5 mg / kg body weight. A particularly preferred dosage is about 0.25 mg / kg body weight. In one example, the enzyme is administered in the range of 0.5 mg / kg to 1 mg / kg body weight. A particularly preferred dosage is about 1 mg / kg body weight. In some embodiments, two doses of 1 mg / kg body weight of enzyme may be administered.

[0046] The amount of enzyme administered is typically sufficient to produce a desired effect, such as extending the half-life of the gene therapy agent and / or reducing the amount of gene therapy agent required to achieve the desired effect. The combined use of an enzyme and a gene therapy agent may produce a synergistic effect. Therefore, the amounts of the enzyme and gene therapy agent may be sufficient to produce such a synergistic effect. The effect may be, for example, a reduction in the amount of gene therapy agent that needs to be administered. Alternatively or additionally, the half-life of the gene therapy agent may be extended. The half-life of the gene therapy agent may be measured by qPCR. One preferred method for measuring serum half-life is to use qPCR to detect the gene therapy agent in the serum and / or plasma of a subject.

[0047] In particularly preferred cases, the amount of enzyme administered is sufficient to degrade or digest all or substantially all IgG molecules present in the subject's serum and / or inhibit or reduce the effector function of the IgG molecules, and optionally the enzyme may be an IgG cysteine ​​protease or an IgG endoglycosidase. Here, "substantially all" can mean at least 90%. Preferably, "substantially all" means at least 95%. Even more preferably, "substantially all" means at least 99%.

[0048] The enzyme may be administered multiple times to the same subject, provided that the amount of anti-drug antibodies (ADA) capable of binding to the enzyme in the subject's serum does not exceed a threshold determined by a clinician. Preferably, the enzyme is administered before the gene therapy agent. For example, the enzyme may be administered 4 to 96 hours before the administration of the gene therapy agent. For example, the enzyme may be administered 4 to 60 hours before the administration of the gene therapy agent. Preferably, the enzyme may be administered 24 to 60 hours before the administration of the gene therapy agent. More preferably, the enzyme may be administered 24 to 48 hours before the administration of the gene therapy agent. In one embodiment, the gene therapy agent is administered within 48 hours after the administration of the enzyme.

[0049] In some embodiments, the enzyme may be administered before and / or after administration of a gene therapy agent. For example, as described above, the enzyme may be administered before administration or within a certain time period after administration of the gene therapy agent. Thus, for example, the enzyme may be administered within 48 hours after administration of the gene therapy agent. The enzyme may be administered within 4 to 72 hours after administration of the gene therapy agent. The enzyme may be administered between 4 and 6 hours after administration of the gene therapy agent.

[0050] In some embodiments, the enzyme and gene therapy agent may be administered cyclically. For example, each cycle may include administration prior to gene therapy at the timings described above, and optionally subsequent administrations. This may be repeated, for example, 2, 3, 4, 5, or more times, in which case administration of the enzyme followed by administration of the gene therapy agent is considered one cycle.

[0051] In some embodiments, the invention may involve measuring ADA in response to a gene therapy agent, or may involve subjects in whom such measurement has already been performed, e.g., an enzyme may be administered until the level of ADA in response to the gene therapy agent falls below a certain threshold.

[0052] The amount of ADA present in the subject's serum and capable of binding to the protease can be measured by any suitable method, such as an agent-specific CAP FEIA (immunoCAP) test or titer assay. If the ADA in the subject exceeds the aforementioned threshold, the treatment regimen may include administration of a replacement enzyme. In one embodiment, the assay used may be a luciferase-based assay. In another embodiment, the assay used may be a bridging assay or a sandwich assay. In yet another embodiment, the assay may be an MSD assay.

[0053] Preferred enzymes include IgG cysteine ​​proteases from Streptococcus bacteria, such as Streptococcus pyogenes, Streptococcus equi, Streptococcus equi subspecies zooepidemicus, or a newly described streptococcus species tentatively designated Streptococcus krosus. Particularly preferred polypeptides include IdeS, MAC2, SpeB, IdeZ, hybrid IdeS / IdeZ (e.g., the enzymes described in WO 2016 / 128558 or WO 2021 / 233911), IgdE, Xork, or the IdE enzyme described in WO 2021 / 254479.

[0054] Further preferred enzymes that can be used include IgG endoglycosidases. Preferred IgG endoglycosidases include those derived from Streptococcus bacteria, such as Streptococcus pyogenes, Streptococcus equi, or Streptococcus zooepidemicus, or Corynebacterium pseudotuberculosis, Enterococcus faecalis, or Elizabethkingia meningoseptica. Particularly preferred enzymes are EndoS, CP40, EndoE, or EndoF2 polypeptides. An especially preferred enzyme is an imfilidase. An even more especially preferred enzyme is EndoS.

[0055] IgG cysteine ​​protease The IgG cysteine ​​protease used in the present invention is typically specific for IgG. In a preferred embodiment, the protease used in the method of the present invention is IdeS ( Immunoglobulin G- d egrading e nzyme of S IdeS is an extracellular cysteine ​​protease produced by the human pathogen S. pyogenes. IdeS was originally isolated from a serotype M1 group A streptococcus strain, but the IdeS gene has now been identified in all group A streptococcus strains tested. IdeS has very high substrate specificity, with IgG being the only identified substrate. IdeS catalyzes a single proteolytic cleavage in the lower hinge region of the heavy chains of all human IgG subclasses. IdeS also catalyzes similar cleavage in the heavy chains of several IgG subclasses in various animal species. IdeS efficiently cleaves IgG into Fc and F(ab')2 fragments via a two-step mechanism. In the first step, the first heavy chain (first heavy chain) of IgG is cleaved to generate a single cleaved IgG (scIgG) molecule with a noncovalently bound Fc / 2 molecule. This scIgG molecule is an intermediate product, retaining the remaining second heavy chain (the ds chain) of the original IgG molecule. In the second step of the mechanism, IdeS cleaves the ds chain, releasing the F(ab')2 fragment and the homodimeric Fc fragment. These are the commonly observed products under physiological conditions. Under reducing conditions, the F(ab')2 fragment dissociates into two Fab' fragments, and the homodimeric Fc fragment can dissociate into its constituent monomers. IdeS has been shown to be particularly effective at cleaving human IgG. Within hours of IdeS administration, the entire plasma IgG pool is cleaved, and circulating IgG levels remain low for approximately one week, after which newly synthesized IgG appears in plasma. This demonstrates that IdeS cleaves not only the plasma pool (i.e., serum IgG molecules) but also the entire extracellular plasma IgG pool (Winstedt et al.; PloS One 2015;10(7):e0132011).

[0056] SEQ ID NO: 1 is the full-length sequence of IdeS, including the N-terminal methionine and signal sequence, and is also available as NCBI reference sequence WP_010922160.1. SEQ ID NO: 2 is the mature sequence of IdeS lacking the N-terminal methionine and signal sequence, and is also available as Genbank accession number ADF13949.1.

[0057] In another embodiment, the protease for use in the methods of the present invention is IdeZ, an IgG cysteine ​​protease produced by Streptococcus equi ssp. Zooepidemicus is a bacterium found primarily in horses. SEQ ID NO: 3 is the full-length sequence of IdeZ, including the N-terminal methionine and signal sequence. It is also available as NCBI reference sequence number WP_014622780.1. SEQ ID NO: 4 is the mature sequence of IdeZ lacking the N-terminal methionine and signal sequence.

[0058] In another embodiment, the protease for use in the methods of the invention is a hybrid IdeS / Z, as shown in the sequence of SEQ ID NO: 5. The N-terminus is based on IdeZ, lacking the N-terminal methionine and signal sequence.

[0059] In a preferred embodiment, the protease used in the present invention may comprise or consist of SEQ ID NO: 2, 4, or 5. The proteases used in the present invention may comprise an additional N-terminal methionine (M) residue and / or a C-terminal tag to aid expression in and isolation from standard bacterial expression systems. Suitable tags include histidine tags, which may be directly linked to the C-terminus of the polypeptide or indirectly linked via any suitable linker sequence, such as 3, 4, or 5 glycine residues. Histidine tags will typically consist of 6 histidine residues, but may be longer, typically up to 7, 8, 9, 10, or 20 amino acids in length, or may be shorter, e.g., 5, 4, 3, 2, or 1 amino acid.

[0060] In a further preferred embodiment, the protease used in the present invention may comprise, consist essentially of, or consist of any one of the sequences set forth in SEQ ID NOs: 6 to 25. These sequences represent IdeS and IdeZ polypeptides with increased protease activity and / or reduced immunogenicity. Each of SEQ ID NOs: 6 to 25 may optionally contain an additional methionine at the N-terminus and / or a histidine tag at the C-terminus. The histidine tag preferably consists of six histidine residues. The histidine tag is preferably linked to the C-terminus via a linker consisting of three or five glycine residues.

[0061] In a further preferred embodiment, the protease used in the present invention may comprise, consist essentially of, or consist of any one of the sequences set forth in SEQ ID NOs: 56 to 69. These sequences represent IdeS polypeptides with increased protease activity and / or reduced immunogenicity. Each of SEQ ID NOs: 56 to 69 may optionally contain an additional methionine at the N-terminus and / or a histidine tag at the C-terminus. The histidine tag preferably consists of six histidine residues. The histidine tag is preferably linked to the C-terminus via a linker of three or five glycine residues.

[0062] In a further preferred embodiment, the proteases for use in the present invention comprise, consist essentially of, or consist of any one of SEQ ID NOs: 6 to 25, optionally containing up to three amino acid substitutions (e.g., one, two, or three residues). Each of SEQ ID NOs: 6 to 25 and variants thereof may optionally contain an additional N-terminal methionine and / or a C-terminal histidine tag.

[0063] In a further preferred embodiment, the proteases for use in the present invention comprise, consist essentially of, or consist of any one of SEQ ID NOs: 56-69, optionally containing up to three (e.g., one, two, or three) amino acid substitutions. Each of SEQ ID NOs: 56-69 and variants thereof may optionally contain an additional N-terminal methionine and / or a C-terminal histidine tag.

[0064] Polypeptides of the present invention are typically at least 100, 150, 200, 250, 260, 270, 280, 290, 300, or 310 amino acids in length. Polypeptides of the present invention are typically no more than 400, 350, 340, 330, 320, or 315 amino acids in length. In providing ranges of polypeptide length of the present invention, it will be recognized that any of the lower limits set forth above may be combined with any of the upper limits set forth above. For example, polypeptides may be 100 to 400 amino acids in length, or alternatively, 250 to 350 amino acids in length. Polypeptides are preferably 290 to 320 amino acids in length, and most preferably 300 to 315 amino acids in length.

[0065] The primary structure (amino acid sequence) of the protease of the present invention is based on the primary structure of IdeS, IdeZ, or IdeS / Z, specifically the amino acid sequence of SEQ ID NO: 2, 4, or 5, respectively. The sequence of the protease of the present invention may include a variant of the amino acid sequence of SEQ ID NO: 2, 4, or 5 that is at least 80% identical to the amino acid sequence of SEQ ID NO: 2, 4, or 5. The variant sequence may be at least 80%, at least 85%, preferably at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequence of SEQ ID NO: 2, 4, or 5. The variant sequence may be identical to the sequence of SEQ ID NO: 2, 4, or 5, except for including one or more of the specific modifications described in WO 2016 / 128558 or WO 2016 / 128559. Identity to the sequence of SEQ ID NO: 2, 4 or 5 may be measured over a region of at least 50, at least 100, at least 200, at least 300 or more consecutive amino acids of the sequence shown in SEQ ID NO: 2, 4 or 5, and more preferably over the entire length of SEQ ID NO: 4 or 5.

[0066] The protease for use in the present invention may be an IdeS, IdeZ, or IdeS / Z polypeptide comprising a variant of the amino acid sequence of SEQ ID NO: 2, 4, or 5, which has been modified, such as by adding, deleting, or substituting amino acids, compared to the sequence of SEQ ID NO: 2, 4, or 5. Such modifications are preferably conservative amino acid substitutions. Conservative substitutions refer to the replacement of an amino acid with an amino acid that has a similar chemical structure, chemical properties, or side chain volume. The introduced amino acids may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino acids they replace. Alternatively, conservative substitutions may involve the introduction of another aromatic amino acid in place of an already existing aromatic amino acid, or another aliphatic amino acid in place of an already existing aliphatic amino acid. Conservative amino acid substitutions are known in the art.

[0067] IgG cysteine ​​protease activity can be assessed by any suitable method, such as incubating a polypeptide with a sample containing IgG and determining the presence of IgG cleavage products. Suitable methods include those described in WO 2016 / 128559. Suitable assays include the ELISA-based assay described in WO 2016 / 128559. In such assays, the wells of an assay plate are typically coated with an antibody target, such as bovine serum albumin (BSA). A sample of the polypeptide to be tested is then added to the well, followed by a sample of the target-specific antibody (in this example, a BSA-specific antibody). The polypeptide and antibody are allowed to react under conditions suitable for IgG cysteine ​​protease activity. After a suitable time, the assay plate is washed, and a detection antibody that specifically binds to the target-specific antibody is added under conditions suitable for binding to the target-specific antibody. The detection antibody may bind to any untreated target-specific antibody that bound to the target in each well. After washing, the amount of detection antibody present in the well may be proportional to the amount of target-specific antibody bound to the well. The detection antibody may be directly or indirectly conjugated to a label or another reporter system (e.g., an enzyme), allowing the amount of detection antibody remaining in each well to be determined. The higher the activity of the polypeptide being tested, the lower the amount of untreated target-specific antibody remaining in the well, and therefore the lower the amount of detection antibody. Typically, at least one well of a given assay plate contains IdeS instead of the polypeptide being tested, allowing the activity of the polypeptide being tested to be directly compared with that of IdeS. IdeZ and IdeS / Z may also be included in the comparison. Another suitable assay is the MSD assay. In this assay, the wells of the assay plate are also typically coated with an antibody target, such as bovine serum albumin (BSA). A sample of the polypeptide being tested is then added to the well, followed by a sample of the target-specific antibody (in this example, a BSA-specific antibody). The polypeptide and antibody are allowed to react under conditions suitable for IgG cysteine ​​protease activity.After an appropriate time, the assay plate is washed, and a detection antibody that specifically binds to the target-specific antibody is reacted under conditions suitable for binding to the target-specific antibody. The detection antibody may bind to any untreated target-specific antibody that bound to the target in each well. After washing, the amount of detection antibody present in each well may be proportional to the amount of target-specific antibody bound to that well. The detection antibody may be directly or indirectly conjugated to a SULFO tag, which emits light when a voltage is applied to the plate electrodes in the device.

[0068] Other assays include those that determine the activity of a test polypeptide by directly visualizing and / or quantifying IgG fragments resulting from IgG cleavage by the test polypeptide. This type of assay is also described in WO 2016 / 128559. In such assays, an IgG sample is typically incubated with the test polypeptide (or one or more of IdeS, IdeZ, and / or IdeS / Z as controls) at different concentrations in a titration series. The products obtained after incubation at each concentration are separated using gel electrophoresis, e.g., SDS-PAGE. Intact IgG and fragments resulting from IgG cleavage can be identified by their size and quantified by the intensity of staining with an appropriate stain. The greater the amount of cleaved fragments, the greater the activity of the test polypeptide at that concentration. The polypeptides of the present invention typically produce detectable amounts of cleaved fragments at lower concentrations (lower points in the titration series) than IdeZ and / or IdeS. This type of assay also allows for the identification of test polypeptides that are more effective at cleaving the first heavy chain or the second heavy chain of IgG molecules. The amount of different fragments resulting from each cleavage event can also be determined. The polypeptides of the present invention may be more effective at cleaving the first heavy chain of IgG molecules than the second chain, particularly when the IgG is of the IgG2 isotype. The polypeptides of the present invention may also be more effective at cleaving IgG1 than IgG2.

[0069] In particularly preferred examples, the IgG cysteine ​​protease is a polypeptide comprising or consisting of a sequence at least 80% identical, at least 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 2, 4 or 5. In even more particularly preferred examples, the IgG cysteine ​​protease comprises or consists of any one of the sequences set forth in SEQ ID NOs: 6 to 25, 55 to 69, 91 or 92. Optionally, the sequence comprises an additional methionine at the N-terminus and / or a histidine tag at the C-terminus.

[0070] IgG endoglycosidase The enzyme may have IgG endoglycosidase activity, and preferably cleaves the sugar chain moiety at Asn297 (Kabat numbering) in the Fc region of IgG. Examples of such proteins include EndoS ( Endo glycosidase of S. EndoS is a Streptococcus pyogenes (Streptococcus pyogenes) strain. EndoS hydrolyzes the β-1,4-di-N-acetylchitobiose core of the asparagine-linked glycan of conventional glycosylated IgG. The mature sequence of EndoS is provided as SEQ ID NO: 90. The agent may be a protein comprising or consisting of the amino acid sequence of SEQ ID NO: 90, or a homolog thereof from another bacterium, such as Streptococcus equi, Streptococcus zooepidemicus, Corynebacterium pseudotuberculosis, Enterococcus faecalis, or Elizabethkingia meningoseptica. The agent may be CP40, EndoE, or EndoF2.

[0071] Alternatively, the agent may be a variant of the EndoS protein comprising or consisting of any amino acid sequence having at least 80%, 85%, 90% or 95% identity to SEQ ID NO: 90 and having IgG endoglycosidase activity. The variant of the EndoS protein may comprise or consist of an amino acid sequence having up to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or more amino acid substitutions, insertions or deletions compared to the amino acid sequence of SEQ ID NO: 90, so long as it has IgG endoglycosidase activity. The amino acid substitutions are preferably conservative. Conservative substitutions are as defined in the previous section.

[0072] Alternatively, the agent may be a protein comprising or consisting of a fragment of SEQ ID NO: 90 and having IgG endoglycosidase activity. Preferably, the fragment is 400 to 950, 500 to 950, 600 to 950, 700 to 950, or 800 to 950 amino acids in length. A preferred fragment consists of amino acids 1 to 409 of SEQ ID NO: 90 and corresponds to the enzymatically active α-domain of EndoS generated by cleavage with the streptococcal cysteine ​​protease SpeB. The fragment may be generated by deleting one or more amino acid residues from the amino acid sequence of SEQ ID NO: 90. Up to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, or 550 residues or more may be deleted. The deleted residues may be contiguous.

[0073] Any fragment or variant of SEQ ID NO:90 preferably comprises residues 191-199 of SEQ ID NO:90, i.e., Leu191, Asp192, Gly193, Leu194, Asp195, Val196, Asp197, Val198, and Glu199 of SEQ ID NO:90. These amino acids constitute the complete chitinase family 18 active site, which ends with glutamic acid. Glutamic acid in the active site of chitinase is essential for enzymatic activity. Therefore, most preferably, a variant of SEQ ID NO:90 comprises Glu199 of SEQ ID NO:90. A variant of SEQ ID NO:90 may comprise residues 191-199 of SEQ ID NO:90 with one or more conservative substitutions, provided that the variant comprises Glu199 of SEQ ID NO:90.

[0074] gene therapy agents The gene therapy agent for use in the present invention may be any suitable gene therapy agent. It may be a gene therapy agent for treating any of the conditions described herein. For example, the gene therapy agent may include a nucleic acid encoding a therapeutic gene of interest and a means for its expression. The therapeutic gene may be, for example, one that can complement a dysfunctional or deleted gene associated with any of the conditions described herein. For example, the gene therapy may be gene enhancement therapy, gene inhibition therapy, or suicide gene therapy. Preferably, it is gene enhancement therapy.

[0075] Preferably, the gene therapy is viral gene therapy. A particularly preferred example of viral gene therapy is AAV (adeno-associated virus) gene therapy. Additional possible examples of gene therapy vectors include adenoviral vectors. Other examples of gene therapy vectors include lentiviral and retroviral gene therapy vectors. Additional examples of gene therapy vectors include CRISPR-based gene therapy vectors.

[0076] In one example, the gene therapy agent comprises a lentiviral vector comprising an envelope protein to which the subject's antibody specifically binds. In a preferred example, the gene therapy agent comprises an AAV vector comprising a capsid protein to which the subject's antibody specifically binds. Optionally, the subject's antibody specifically binds to the VP1, VP2, and / or VP3 capsid proteins.

[0077] Examples of AAV vectors that can be utilized include, for example, AAV type 1, AAV type 2, AAV type 3 (e.g., types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAV type rh32.33, AAV type rh8, AAV type rhlO, AAV type rh74, AAV type hu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AV2i8, AAV2g9, AAV-LK03, AAV7m8, AAVAnc80, AAVPHP.B, and any other AAV now known or hereafter discovered. See, for example, Bernard N. Fields et al., VIROLOGY, volume 2, chapter 69 (4th ed., Lippincott-Raven Publishers). Numerous AAV serotypes and strains have been identified (see, for example, Gao et al., (2004) J. Virology 78:6381-6388; Morris et al., (2004) Virology 33:375-383). The AAV utilized may be a chimeric AAV, i.e., an AAV having a capsid protein comprising regions, domains, or individual amino acids from two or more different serotypes. AAV vectors may also be developed by directed evolution methods.

[0078] In one aspect, the AAV vector comprises a VP1, VP2 and / or VP3 capsid protein that has at least 60%, 70%, 80%, 90%, 95% or 100% sequence identity to a VP1, VP2 and / or VP3 capsid protein selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV3B, AAV-2i8, RhlO, Rh74, SEQ ID NO:93 and SEQ ID NO:94.

[0079] The gene therapy agent may be administered at an appropriate dose. One advantage of the present invention is that the use of an enzyme generally reduces the amount of gene therapy agent to be administered compared to when the enzyme is not used. Thus, the enzyme and the gene therapy agent form a synergistic combination. Examples of reductions include a decrease of at least 5%. In one example, the reduction is at least 10%. In another example, the reduction is at least 20%. For example, in some examples, the reduction is at least 25%, 30%, 40%, at least 50%, or more. The reduction is compared to the dose normally administered. In another example, the reduction is within a range formed by any two of these values. In another embodiment, the same dose is administered rather than a reduced dose, but the effect of gene therapy may be greater, for example, the expression level may increase by more than any of the percentage values ​​mentioned in this paragraph.

[0080] The amount of gene therapy agent typically administered in the absence of enzymes is well known to those skilled in the art. For example, if a gene therapy agent has received marketing approval, the dosage is described in the package insert and / or the Summary of Product Characteristics list available from the regulatory agency. Additionally or alternatively, the amount may be any amount commonly known in the art. For example, different AAV serotypes (AAV1, AAV2, AAV5, AAV8, and AAV9) are administered at doses of 2 x 10 per kg body weight per patient in gene therapy for these inherited diseases. 11 ~2×10 14Vector doses in the range of vector genomes (vgs / kg) have been used (Maurya et al.; Cancer Gene Ther. 2022 Oct;29(10):1305-1306). Of course, the amount typically administered is assessed in the same species, e.g., humans, if the subject is a human.

[0081] In a preferred embodiment, the gene therapy agent is a viral vector and is administered in an amount of 1×10 15 In some instances, the vector is administered at a dose lower than 10 vector genomes (vg) / kg body weight (vg / kg). 14 In some instances, the dose may be less than 10 vg / kg body weight. 13 In some preferred embodiments, the dose may be less than 10 vg / kg body weight. 12 In some more preferred embodiments, the dose may be less than 10 vg / kg body weight. 11 In some more preferred embodiments, the dose may be less than 1×10 vg / kg body weight. 10 vg / kg, less than 1×10 9 vg / kg, less than 1×10 8 vg / kg or less than 0.99 x 10 7 The dose may be lower than 1000 mg / kg. The present invention can induce a reduction in the dose to be administered to one of these levels. Furthermore, the present invention may make it possible to reduce the administered dose to a level that allows gene therapy to be applied to subjects who have not previously been considered candidates for gene therapy, while maintaining efficacy.

[0082] If the gene therapy is a viral vector, typically 2 × 10 11 From 2x10 14 For example, the approved gene therapy vector treatments Elevidys™ and Zolgensma™ are administered at doses between 1.33 x 10 13 vg / mL and 1.1x10 14The dose of gene therapy agents typically administered (i.e., when step a described in the present invention is not performed) is well known to those skilled in the art and can also be found in published documents from regulatory authorities, such as the Summary of Product Characteristics. According to the present invention, when step a is not performed, a subject is administered 2×10 11 ~2×10 14 The gene therapy agent administered at a dose ranging from 2 × 10 11 (e.g., 1×10 11 , 2 × 10 10 , 1×10 11 , 2 × 10 9 or 1 x 10 9 ) can be administered at a dose lower than 2×10 11 (e.g., 1×10 11 , 2 × 10 10 , 1×10 11 , 2 × 10 9 or 1 x 10 9 ), which is lower than the 2×10 dose when step a is not performed. 11 ~2×10 14 The dose may be administered in the range of

[0083] In another embodiment, the gene therapy agent may be a non-viral vector, for example, the gene therapy agent may be selected from the group consisting of a liposome, a nanoparticle, a lipid nanoparticle, a polymer, a microparticle, a microcapsule, a micelle, or an extracellular vesicle.

[0084] The present invention may induce an increase in a subject's exposure to a gene therapy agent. The duration of exposure may be extended. The amount of exposure at a particular time point may be greater using the present invention. The overall amount of exposure (both in level and duration) may be increased. The present invention may induce an increase in the level of gene therapy agent reaching a certain target organ. The overall duration of exposure to the gene therapy agent may be extended. Such an increase may be, for example, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, or at least 100%. The increase may also be within a range between the endpoints of these values.

[0085] The present invention provides uses for achieving any of the effects described herein using the enzymes described herein, wherein a gene therapy agent is also administered to the subject.

[0086] Diseases and Conditions to be Treated Gene therapy can potentially be used to treat almost any disease or disorder.

[0087] In a preferred embodiment, the disease to be treated is caused by a loss of protein function or activity, and the gene therapy agent comprises a heterologous polynucleotide that encodes a protein or peptide that, when expressed in a subject, confers or complements the function or activity of the protein. In a further preferred embodiment, the disease to be treated is caused by a gain of protein function, activity, or expression, and the gene therapy agent comprises a heterologous polynucleotide that is transcribed into a nucleic acid that inhibits, reduces, or decreases the expression of the function, activity, or gain of expression of the protein.

[0088] Preferred diseases and disorders that can be treated using the present invention include proliferative diseases (cancer, tumors, dysplasia, etc.), metabolic diseases such as Crigler-Najjar syndrome and metabolic diseases of the liver, Friedreich's ataxia, infectious diseases, addictions (e.g., to tobacco, alcohol, or drugs), epilepsy, Canavan disease, adrenoleukodystrophy, viral diseases (including, e.g., hepatitis B or C virus, HIV, herpes, retroviruses, etc.), genetic diseases (cystic fibrosis, dystroglycanopathy, muscle diseases such as Duchenne muscular dystrophy or dystrophies, myotubular myopathy, hemophilia A, hemophilia B, sickle cell anemia, sickle cell disease, Fanconi anemia, diabetes, amyotrophic lateral sclerosis (ALS), myotubulin myopathy, spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy, or Charcot-Mansfield disease. These conditions include motor neuron diseases such as Lea-Tooth disease, arthritis, severe combined immunodeficiency disorders (such as RS-SCID, ADA-SCID, or X-SCID), Wiskott-Aldrich syndrome, X-linked thrombocytopenia, X-linked congenital neutropenia, and chronic granulomatous disease, clotting factor deficiencies, cardiovascular diseases (such as restenosis, ischemia, dyslipidemia, and homozygous familial hypercholesterolemia), eye diseases such as retinitis pigmentosa, Leber's congenital amaurosis, Leber's hereditary optic neuropathy, and Stargardt disease, lysosomal storage diseases such as Sanfilippo syndrome, hyperbilirubinaemias such as CN1 or CN2 or Gilbert's syndrome, and glycogen storage diseases such as Fabry disease, GSD1, GSD2 (Pompe disease), GSD3, GSD4, GSD5, GSD6, GSD7, GSD8, GSD9, GSD10, GSD11, GSD12, and fatal congenital glycogen storage diseases of the heart.

[0089] In one embodiment, the subject to be treated has any of the following diseases or conditions: Pulmonary diseases (e.g., cystic fibrosis); bleeding disorders (e.g., hemophilia A or hemophilia B (with or without inhibitors)), thalassemia, blood disorders (e.g., anemia), Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), epilepsy, lysosomal storage diseases (e.g., aspartylglucosaminuria), Batten disease, late-onset infantile neuronal ceroid lipofuscinosis type II (CLN2), cystinosis, Fabry disease, Gaucher disease types I, II, and III, glycogen storage disease type II (Pompe disease), GM2-gangliosidosis type I (Tay-Sachs disease), GM2-gangliosidosis type II (Sandhoff disease), mucolipidosis type 1 (sialidosis types I and II), type II ( I-cell disease), type III (pseudo-Hurler disease) and type IV, mucopolysaccharidosis (Hurler disease and variants, Hunter, Sanfilippo types A, B, C, D, Morquio types A and B, Marote-Lamy and Sly disease), Niemann-Pick disease types A / B, C1 and C2, and Schindler disease types I and II), hereditary angioedema (HAE), copper or iron storage disorders (e.g., Wilson disease or Menkes disease), lysosomal acid lipase deficiency, neuropathic or neurodegenerative disorders, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, metabolic deficiencies (e.g., glycogen storage diseases), solid organ diseases (e.g., brain, liver, kidney, heart), or viral infections (e.g., hepatitis B and C, HIV, etc.), bacterial or fungal diseases; blood clotting disorders, optionally hemophilia A, hemophilia A with inhibitor antibodies, hemophilia B, hemophilia B with inhibitor antibodies, deficiency of any of the following clotting factors: factor VII, factor VIII, factor IX, factor X, factor XI, factor V, factor XII, factor II, von Willebrand factor, or combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase C1 deficiency, or gamma-carboxylase deficiency; Anemia, bleeding associated with trauma or injury, thrombosis, thrombocytopenia, stroke, coagulopathy, diffuse intravascular coagulation (DIC); excessive anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotic agents (i.e., FXa inhibitors), or thrombocytopenia such as Bernard-Soulier syndrome, Glanzmann thrombasthenia, or storage pool deficiency.

[0090] In another preferred embodiment, the gene therapy agent comprises an inhibitory nucleic acid that binds to a gene, a transcript of the gene, or a transcript of the gene associated with a polynucleotide repeat disease selected from the group consisting of the Huntington (HTT) gene, a gene associated with dentatorubral-pallidoluysian atrophy (atrophin 1, ATN1), the androgen receptor gene on the X chromosome in spinal-bulbar muscular atrophy, human Ataxin-1, -2, -3, and -7, Cav2.1 P / Q voltage-gated calcium channel (CACNA1A), TATA-binding protein, Ataxin 8 reverse chain (ATXN80S), serine / threonine protein phosphatase 2A 55 kDa regulatory subunit B beta isoform in spinocerebellar degeneration (types 1, 2, 3, 6, 7, 8, 12, 17), FMR1 (fragile X mental retardation 1) in fragile X syndrome, and FMR1 (fragile X associated tremor / ataxia syndrome) in fragile X syndrome. fragile X mental retardation 1, FMR1 (fragile X mental retardation 2) or AF4 / FMR2 family member 2 in fragile XE mental retardation; myotonin-protein kinase (MT-PK) in myotonic dystrophy; frataxin in Friedreich's ataxia; mutant superoxide dismutase 1 (SOD1) genes in amyotrophic lateral sclerosis; genes involved in the pathogenesis of Parkinson's disease and / or Alzheimer's disease; apolipoprotein B (APOB) and proprotein convertase subtilisin / kexin type 9 (PCSK9) in hypercholesterolemia; HIV Tat, human immunodeficiency virus transactivator response element of the transcription gene in HIV infection; HIV TAR, HIV TAR, human immunodeficiency virus transcriptional activator human immunodeficiency virus transactivator response element gene; CC chemokine receptor (CCR5) in HIV infection;Rous sarcoma virus (RSV) nucleocapsid protein in RSV infection, liver-specific microRNA (miR-l22) in hepatitis C virus infection; p53 in acute renal failure, delayed graft function, or renal failure; protein kinase N3 (PKN3) in highly recurrent or metastatic solid malignancies; LMP2 (also known as proteasome beta subunit 9 (PSMB9)) in malignant melanoma; LMP7 (also known as proteasome beta subunit 9 (PSMB8)) in malignant melanoma; MECL1 (also known as proteasome beta subunit 10 (PSMB10)) in malignant melanoma; vascular endothelial growth factor (VEGF) in solid tumors; kinesin spindle protein in solid tumors; and apoptosis inhibitor B-cell in chronic myeloid leukemia. Ribonucleotide reductase M2 (RRM2) in solid tumors; Furin in solid tumors; polo-like kinase 1 (PLK1) in hepatocellular carcinoma; diacylglycerol acyltransferase 1 (DGAT1) in hepatitis C infection; beta-catenin in familial adenomatous polyposis; beta-2 adrenergic receptor in glaucoma; RTP80l / Reddl (also known as DNA damage-induced transcript 4 protein) in diabetic macular edema (DME) or age-related macular degeneration; vascular endothelial growth factor receptor I (VEGFR1) in age-related macular degeneration or choroidal neovascularization; and caspases in non-arteritic ischemic optic neuropathy. 2; keratin 6AN17K mutant protein in congenital pachyonychia; influenza A virus genome / gene sequence in influenza infection; SARS coronavirus genome / gene sequence in severe acute respiratory syndrome (SARS) infection; respiratory syncytial virus genome / gene sequence in respiratory syncytial virus infection; Ebola virus genome / gene sequence in Ebola hemorrhagic fever infection; hepatitis B and C virus genome / gene sequence in hepatitis B and C infection;Herpes simplex virus (HSV) genome / gene sequences in HSV infections, Coxsackievirus B3 genome / gene sequences in Coxsackievirus B3 infections; silencing of pathogenic alleles of torsinA (TOR1A)-like genes (allele-specific silencing) in primary dystonia, pan-class I and HLA allele-specific in transplantation; and mutant rhodopsin gene (RHO) inhibitory nucleic acids in autosomal dominant retinitis pigmentosa (adRP); wherein the inhibitory nucleic acid is optionally an siRNA, antisense molecule, miRNA, RNAi, ribozyme, or shRNA;

[0091] The gene therapy agent may induce the expression of a protein that is missing or deficient in a subject. For example, the present invention may be used to treat growth hormone deficiency. It may also be used to treat immunodeficiency. In another example, the gene therapy agent may induce the killing of cells involved in the pathology underlying the disease state. Further examples of diseases that may be treated include sickle cell disease, SCID, CF, hemophilia, DMD, Huntington's disease, Parkinson's disease, hypercholesterolemia, alpha-trypsin deficiency, CGD, Faconi anemia, and Gaucher disease.

[0092] Gene therapy is typically a treatment method in which a gene therapeutic agent is directly administered to a subject. Treating the disorders or conditions identified herein includes both prevention and treatment. Treating may also include alleviating or ameliorating one or more symptoms of the disorder or disease as a whole.

[0093] The present invention also provides an enzyme that inactivates serum antibodies for use in a method for preventing or treating the above-mentioned diseases or conditions. The present invention also provides use of an enzyme that inactivates serum antibodies in a subject in the manufacture of a medicament, the medicament being for preventing or treating the disease or condition in the above-mentioned method.

[0094] The present invention also provides a gene therapy agent for use in a method for the prevention or treatment of the above-mentioned diseases or conditions. The present invention also provides a gene therapy agent in the manufacture of a medicament, wherein the medicament is for the prevention or treatment of a disease or condition in the above-mentioned method.

[0095] Treatment target The subject to be treated may typically have any of the conditions described herein. One advantage of the present invention is that it is effective even in subjects who do not have antibodies to the gene therapy agent or who have only relatively low levels of such antibodies. Thus, in one example, the subject has no detectable antibodies that specifically bind to the gene therapy agent. In another example, the subject may have a low titer of neutralizing antibodies (NAbs) that specifically bind to the gene therapy agent. Neutralizing antibodies may, for example, prevent the gene therapy agent from binding to its target or target cells. In one embodiment, the subject may have an NAb titer of less than 1:10 or a TAb titer of less than 1:100. The NAb titer may be less than 1:50, less than 1:100, less than 1:500, or less than 1:1000. The TAb titer may be less than 1:500, less than 1:1000, less than 1:10,000, or even lower.

[0096] Since the effect of gene therapy is enhanced by the present invention, the present invention can be applied to subjects who have not been candidates for gene therapy in the past due to insufficient effects of gene therapy.

[0097] In other embodiments, the present invention may be applied not only to specific patient groups, but also to patients in general. Because the present invention contributes to improving gene therapy, it may be applicable to all patients who would benefit from gene therapy. For example, the ability of the present invention to extend a subject's exposure to a gene therapy agent may be used in gene therapy patients in general, not just those with low or no serum antibodies.

[0098] The subject is typically a mammal, and in one particularly preferred embodiment, the subject is human.

[0099] In one example, the subject may have never received gene therapy before. The subject may also have never received the gene therapy agent that is to be administered as part of the method.

[0100] Compositions and formulations comprising polypeptides The present invention also provides compositions comprising enzymes for use in the methods of the present invention. For example, the present invention provides compositions comprising one or more polypeptides and at least one pharmaceutically acceptable carrier or diluent. The present invention also provides compositions comprising gene therapy agents for use in the methods of the present invention. For example, the present invention provides compositions comprising a gene therapy agent and at least one pharmaceutically acceptable carrier or diluent.

[0101] The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not harmful to the subject to which the composition is administered. Typically, the carriers and the ultimate composition are sterile and pyrogen-free.

[0102] Suitable compositions can be formulated using standard pharmaceutical formulation chemistry techniques and methodologies, all of which are readily available to those skilled in the art. For example, the enzyme can be combined with one or more pharmaceutically acceptable excipients or vehicles. Auxiliary substances, such as wetting agents, emulsifiers, pH buffering substances, and reducing agents, may be present in the excipient or vehicle. Examples of suitable reducing agents include cysteine, thioglycerol, thioredoxin, glutathione, and the like. Excipients, vehicles, and auxiliary substances are generally agents that do not induce an immune response in the subject receiving the composition and can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, polyethylene glycol, hyaluronic acid, glycerol, thioglycerol, and ethanol. Pharmaceutically acceptable salts can also be included, such as mineral acid salts such as hydrochlorides, hydrobromides, phosphates, and sulfates; and salts of organic acids such as acetic acid, propionic acid, malonic acid, and benzoic acid. A thorough discussion of pharmaceutically acceptable excipients, vehicles and auxiliary substances can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991).

[0103] Such compositions may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable compositions may be prepared, packaged, or sold in unit-dose form, for example, in ampoules or multidose containers containing a preservative. Compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release formulations or biodegradable agents. Such compositions may further comprise one or more additional ingredients, such as, but not limited to, suspending agents, stabilizers, or dispersing agents. In one embodiment of a composition for parenteral administration, the active ingredient is provided in dry form (e.g., powder or granules) and is reconstituted with a suitable vehicle (e.g., sterile pyrogen-free water) immediately prior to parenteral administration. The composition may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution is formulated according to known techniques and may contain, in addition to the active ingredient, additional ingredients such as dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally acceptable diluent or solvent, such as water or 1,3-butanediol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or diglycerides.

[0104] Other useful parenterally administrable compositions include those containing the active ingredient in microcrystalline form, as a liposomal formulation, or as a component of a biodegradable polymer system. Sustained-release or implant compositions may comprise pharmaceutically acceptable polymeric or hydrophobic materials, such as emulsions, ion-exchange resins, sparingly soluble polymers, or sparingly soluble salts. The compositions may be suitable for administration by any suitable route, including, for example, intradermal, subcutaneous, transdermal, intramuscular, intraarterial, intraperitoneal, intraarticular, intraosseous, or other suitable routes of administration. Preferred compositions are suitable for administration by intravenous injection.

[0105] The compositions of the invention typically provide an "effective amount" or "sufficient amount," where "effective amount" or "sufficient amount" refers to an amount that, administered in single or multiple doses, alone or in combination with one or more other compositions, treatments, protocols, or therapeutic regimens, produces a detectable response, expected or desired result, or benefit in a subject over any period of time (long or short term), to a measurable or detectable extent, or for any period of time (e.g., minutes, hours, days, months, years, or leading to a cure). An "effective amount" or "sufficient amount" for treatment (e.g., an amount to produce symptomatic relief, therapeutic effect, or improvement) is typically an amount effective to produce a measurable response against one, several, or all adverse symptoms, effects, or complications of a disease, or against one or more, several, or all associated adverse symptoms, effects, or complications resulting from or associated with a disease. A satisfactory treatment outcome also occurs when the progression or worsening of a disease is prevented, reduced, inhibited, suppressed, limited, or controlled.

[0106] kit The present invention also provides kits for carrying out the methods described herein. The kits of the present invention may include the enzymes described above or compositions containing the enzymes. The kits may also include means for administering the enzymes or compositions to a subject. The kits may also alternatively or additionally include the gene therapy agents described above or compositions containing same. The kits may also include means for delivering the gene therapy agents. The kits may also include instructions for use of each component in any of the methods described herein.

[0107] Further preferred embodiments The following embodiments are further preferred embodiments, but do not currently form part of the claims, and may be included in the claims in the future. [1] A method for treating a subject in need of gene therapy for a disease, comprising the steps of: a. administering to the subject an enzyme in an amount effective to degrade or digest serum antibodies and / or inhibit or reduce effector functions of serum antibodies in the subject; and b. administering to said subject a gene therapy agent; wherein the gene therapy is administered at a dose lower than the minimum effective dose (MED) that would be administered to the subject in the absence of step a; the subject has either no detectable antibodies that specifically bind to the gene therapy agent, or a low titer of neutralizing antibodies (NAb) that specifically bind to the gene therapy agent, or a low titer of total antibodies (TAb) that specifically bind to the gene therapy agent, and optionally the presence / titer of antibodies in the subject is measured in a serum sample obtained from the subject prior to step a; Optionally, the gene therapy agent is less than 2×10 in the absence of step a. 11 ~2×10 14 2×10 11 The method is administered at a dose lower than that of the [2] The method of embodiment [1], wherein the subject has an NAb titer of less than 1:10 or a TAb titer of less than 1:100. [3] The method according to embodiment [1] or [2], Step a is carried out within a range of about 4 hours to about 48 hours, preferably within a range of about 24 hours to about 48 hours, preferably within a range of about 4 hours to about 24 hours, preferably within a range of about 4 hours to about 6 hours, before step b; and / or Step a and / or step b are performed two or more times; and / or The method, wherein the subject is a human. [4] The method according to any one of embodiments [1] to [3], The method of claim 1, wherein the dose of the enzyme administered in step a is sufficient to degrade or digest all or substantially all IgG molecules present in the serum of the subject and / or inhibit or reduce the effector function of all or substantially all IgG molecules, and optionally the enzyme is an IgG cysteine ​​protease or an IgG endoglycosidase. [5] The method according to embodiment [4], (i) The IgG cysteine protease is derived from Streptococcus, such as Streptococcus pyogenes, Streptococcus equi or Streptococcus zooepidemicus, and optionally, the enzyme is an IdeS, MAC2, SpeB, IdeZ or IdeE polypeptide, or (ii) The IgG endoglycosidase is derived from Streptococcus, such as Streptococcus pyogenes, Streptococcus equi or Streptococcus zooepidemicus, or is derived from Corynebacterium pseudotuberculosis, Enterococcus faecalis or Elizabethkingia meningoseptica, and optionally, the enzyme is an EndoS, CP40, EndoE or EndoF2 polypeptide, method. [6] The method according to embodiment [4] or [5], The IgG cysteine protease is a polypeptide comprising or consisting of a sequence that is at least 80% identical, such as at least 85%, 90%, 95%, 99% or 100% identical, to SEQ ID NO: 2, 4, 5, or the IgG cysteine protease comprises or consists of any one of the sequences of SEQ ID NOs: 6 - 25 and 55 - 69, 91 or 92, and optionally, the sequence contains an additional methionine at the N-terminus and / or a histidine tag at the C-terminus; or The IgG endoglycosidase is a polypeptide comprising or consisting of a sequence that is at least 80% identical, such as at least 85%, 90%, 95%, 99% or 100% identical, to SEQ ID NO: 90, and optionally, the sequence contains an additional methionine at the N-terminus and / or a histidine tag at the C-terminus, method. [7] The method according to any one of embodiments [1] - [6], wherein the enzyme is Imucalase and / or EndoS. [8] The method according to any one of embodiments [1] - [7], wherein the dosage of the enzyme is in the range of 0.25 mg / kg body weight to 0.5 mg / kg body weight, preferably in the range of 0.3 mg / kg body weight to 0.5 mg / kg body weight. [9] The method according to any one of embodiments [1] to [8], wherein the gene therapy agent comprises a viral vector, and optionally comprises a lentiviral vector, an adenoviral vector, or an adeno-associated viral (AAV) vector.

[10] The viral vector is 2×10 11 Vector genome (vg) / kg body weight (vg / kg), 1 × 10 10 vg / kg, less than 1×10 9 vg / kg, less than 1×10 8 vg / kg or less than 0.99x10 7 The method of embodiment [9], wherein the compound is administered at a lower dose.

[11] The method according to embodiment [9] or

[10] , The gene therapy comprises a lentiviral vector comprising an envelope protein to which the subject's antibody specifically binds; or The method, wherein the gene therapy comprises an AAV vector comprising a capsid protein to which the subject's antibody specifically binds, and optionally comprising VP1, VP2 and / or VP3 capsid proteins.

[12] The method of embodiment

[11] , wherein the AAV vector comprises VP1, VP2 and / or VP3 capsid proteins having at least 60%, 70%, 80%, 90%, 95% or 100% sequence identity to a VP1, VP2 and / or VP3 capsid protein selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV3B, AAV-2i8, RhlO, Rh74, VP1, VP2 and / or VP3 capsid proteins of SEQ ID NO:93 and SEQ ID NO:94.

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

[12] , wherein the disease is: (i) caused by a loss of protein function or activity, and the gene therapy agent comprises a heterologous polynucleotide encoding a protein or peptide that, when expressed in the subject, provides or complements the function or activity of the protein; or (ii) A method caused by the gain of a protein function, activity or expression, and wherein the gene therapy agent comprises a heterologous polynucleotide that is transcribed into a nucleic acid that inhibits, reduces or decreases the expression of the gain of the protein function, activity or expression.

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

[13] , wherein the disease is selected from the group consisting of: proliferative diseases (cancer, tumor, dysplasia, etc.), metabolic diseases such as Crigler-Najjar syndrome and metabolic diseases of the liver, Friedreich's ataxia, infectious diseases, poisoning (e.g., to tobacco, alcohol, or drugs), epilepsy, Canavan disease, adrenoleukodystrophy, viral diseases (e.g., hepatitis B or hepatitis C virus), viruses, HIV, herpes, retroviruses, etc.), genetic disorders (cystic fibrosis, dystroglycanopathy, Duchenne muscular dystrophy or muscle diseases such as dystrophies, myotubular myopathy, hemophilia A, hemophilia B, sickle cell anemia, sickle cell disease, Fanconi anemia, diabetes, amyotrophic lateral sclerosis (ALS), myotubulin myopathy, spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy or Charcot-Marie-Tu Motor neuron diseases such as leukemia, arthritis, severe combined immunodeficiency (RS-SCID, ADA-SCID, X-SCID, etc.), Wiskott-Aldrich syndrome, X-linked thrombocytopenia, X-linked congenital neutropenia, chronic granulomatous disease, etc.), coagulation factor deficiency, cardiovascular disease (restenosis, ischemia, dyslipidemia, homozygous familial hypercholesterolemia, etc.), retinitis pigmentosa, Leber's congenital amaurosis, Leber's hereditary optic neuropathy, and Stargall syndrome. lysosomal storage diseases such as Sanfilippo syndrome; hyperbilirubinemia such as CN1 or CN2 or Gilbert's syndrome; glycogen storage diseases such as Fabry disease, GSD1, GSD2 (Pompe disease), GSD3, GSD4, GSD5, GSD6, GSD7, GSD8, GSD9, GSD10, GSD11, GSD12, GSD13, GSD14, GSD15, GSD16, GSD17, GSD18, GSD19, GSD10A, GSD11B, GSD11C, GSD12D, GSD13E, GSD14F, GSD15I, GSD16I, GSD17III, and fatal congenital glycogen storage disease of the heart; and optionally, wherein the vector comprises a therapeutic polynucleotide suitable for treating the disease.

[15] The method according to embodiment

[13] (i) or

[14] , The method, wherein the subject has one of the following diseases: Pulmonary diseases (e.g., cystic fibrosis); bleeding disorders (e.g., hemophilia A or hemophilia B (with or without inhibitors)), thalassemia, blood disorders (e.g., anemia), Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), epilepsy, lysosomal storage diseases (e.g., aspartylglucosaminuria), Batten disease, late-onset infantile neuronal ceroid lipofuscinosis type II (CLN2), cystinosis, Fabry disease, Gaucher disease types I, II, and III, glycogen storage disease type II (Pompe disease), GM2-gangliosidosis type I (Tay-Sachs disease), GM2-gangliosidosis type II (Sandhoff disease), mucolipidosis type 1 (sialidosis types I and II), type II ( I-cell disease), type III (pseudo-Hurler disease) and type IV, mucopolysaccharidosis (Hurler disease and variants, Hunter, Sanfilippo types A, B, C, D, Morquio types A and B, Marote-Lamy and Sly disease), Niemann-Pick disease types A / B, C1 and C2, and Schindler disease types I and II), hereditary angioedema (HAE), copper or iron storage disorders (e.g., Wilson disease or Menkes disease), lysosomal acid lipase deficiency, neuropathic or neurodegenerative disorders, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, metabolic deficiencies (e.g., glycogen storage diseases), solid organ diseases (e.g., brain, liver, kidney, heart), or viral infections (e.g., hepatitis B and C, HIV, etc.), bacterial or fungal diseases; blood clotting disorders, optionally hemophilia A, hemophilia A with inhibitor antibodies, hemophilia B, hemophilia B with inhibitor antibodies, deficiency of any of the following clotting factors: factor VII, factor VIII, factor IX, factor X, factor XI, factor V, factor XII, factor II, von Willebrand factor, or combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase C1 deficiency, or gamma-carboxylase deficiency; Anemia, bleeding associated with trauma or injury, thrombosis, thrombocytopenia, stroke, coagulopathy, diffuse intravascular coagulation (DIC); excessive anticoagulation associated with heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotic agents (i.e., FXa inhibitors), or thrombocytopenia such as Bernard-Soulier syndrome, Glanzmann thrombasthenia, or storage pool deficiency.

[16] The method according to any one of embodiments [1] to

[12] ,

[13] (i),

[14] or

[15] , wherein the heterologous polynucleotide encodes: A protein selected from the group consisting of insulin, glucagon, growth hormone (GH), parathormone (PTH), growth hormone-releasing hormone (GRF), follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony-stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), and tumor cells. growth factor alpha (TGFa), platelet-derived growth factor (PDGF), insulin-like growth factors I and II (IGF-I and IGF-II), TGF, activin, inhibin, bone morphogenetic proteins (BMPs), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neuturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog, and tyrosine hydroxylase; or a protein selected from the group consisting of thrombopoietin (TPO), interleukins (IL-1 to IL-36), monocyte chemotactic proteins, leukemia inhibitory factor, granulocyte colony-stimulating factor, Fas ligand, tumor necrosis factors a and b, interferons a, b and g, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single-chain antibodies, T cell receptors, chimeric T cell receptors, single-chain T cell receptors, MHC class I and class II molecules; or CFTR (cystic fibrosis membrane regulatory protein), a blood coagulation (clotting) factor (factor XIII, factor IX, factor VIII, factor X, factor VII, factor Vila, protein C, etc.), gain-of-function blood coagulation factor, antibody, retinal pigment epithelium-specific 65 kDa protein protein) (RPE65), erythropoietin, FDF receptor, lipoprotein lipase, ornithine transcarbamylase, b-globin, a-globin, spectrin, a-antitrypsin, adenosine deaminase (ADA), metal transporter (ATP7A or ATP7), sulfamidase, lysosomal storage disease-related enzyme (ARSA), hypoxanthine guanine phosphoribosyltransferase, b-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain ketoacid dehydrogenase, hormones, growth factors, insulin-like growth factor 1 or 2, platelet-derived growth factor, epidermal growth factor, nerve growth factor , neurotrophic factor-3 and -4, brain-derived neurotrophic factor, glial cell line-derived growth factor, transforming growth factor a and b, cytokines, α-interferon, β-interferon, interferon-g, interleukin-2, interleukin-4, interleukin-12, granulocyte-macrophage colony-stimulating factor, lymphotoxin, suicide gene products, herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, drug resistance proteins, tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, von Hippel-Lindau (VHF), adenomatous polyposis colicoli (APC)), peptides with immunomodulatory properties, tolerogenic or immunogenic peptides or proteins, Tregitope or hCDRl, insulin, glucokinase, guanylate cyclase 2D (FCA-GUCY2D), Rab escort protein 1 (choroideremia), FCA5 (FCA-Febercilin), ornithine ketoacid aminotransferase (gyrate atrophy), Retinoschisin 1 (X-linked retinoschisis), USH1C (Usher's syndrome 1C), X-linked retinitis pigmentosa GTPase (XFRP), MERTK (AR form of RP: retinitis pigmentosa), DFNB1 (Connexin 26 deafness), ACHM2, 3 and 4 (Achromatopsia), PKD-1 or PKD-2 (Polycystic kidney disease), TPP1, CFN2, sulfotransferase, N-acetylglucosamine-L-phosphotransferase, cathepsin A, GM2-AP, NPC1, VPC2, sphingolipid activator protein, one or more zinc finger nucleases for genome editing, and donor sequences used as one or more repair templates for genome editing; or A gene-editing nuclease, optionally a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN) or a functional type II CRISPR-Cas9.

[17] The method according to any one of embodiments [1] to

[12] ,

[13] (ii), or

[14] , wherein the inhibitory nucleic acid binds to a gene, a transcript of the gene, or a transcript of the gene associated with a polynucleotide repeat disease selected from the group consisting of: Huntington (HTT) gene, genes related to dentatorubral-pallidoluysian atrophy (atrophin1, ATN1), androgen receptor gene on the X chromosome in spinal-bulbar muscular atrophy, human Ataxin-1, -2, -3, and -7, Cav2.1 P / Q voltage-gated calcium channel (CACNA1A), TATA-binding protein, Ataxin 8 reverse chain (ATXN80S), serine / threonine protein phosphatase 2A 55 kDa regulatory subunit B beta isoform in spinocerebellar degeneration (types 1, 2, 3, 6, 7, 8, 12, and 17), FMR1 (fragile X mental retardation 1) in fragile X syndrome, FMR1 (fragile X mental retardation 1) in fragile X-associated tremor / ataxia syndrome, FMR1 (fragile X mental retardation 2) or AF4 / FMR2 in fragile XE mental retardation. Family member 2; myotonin-protein kinase (MT-PK) in myotonic dystrophy; frataxin in Friedreich's ataxia; mutant superoxide dismutase 1 (SOD1) genes in amyotrophic lateral sclerosis; genes involved in the pathogenesis of Parkinson's disease and / or Alzheimer's disease; apolipoprotein B (APOB) and proprotein convertase subtilisin / kexin type 9 (PCSK9) in hypercholesterolemia; HIV Tat, human immunodeficiency virus transactivator response element of transcription genes in HIV infection; HIV TAR, HIV TAR, human immunodeficiency virus transcription activator (human immunodeficiency virus transactivator response element gene); CC chemokine receptor (CCR5) in HIV infection; Rous sarcoma virus (RSV) nucleocapsid protein in RSV infection; liver-specific microRNA (miR-l22) in hepatitis C virus infection;p53 in acute renal failure, delayed graft function, or acute renal failure; protein kinase N3 (PKN3) in highly recurrent or metastatic solid malignancies; LMP2 (LMP2 is also known as proteasome beta subunit 9 (PSMB9)) in malignant melanoma; LMP7 (LMP7 is also known as proteasome beta subunit 9 (PSMB8)) in malignant melanoma; MECL1 (MECL1 is also known as proteasome beta subunit 10 (PSMB10)) in malignant melanoma; vascular endothelial growth factor (VEGF) in solid tumors; kinesin spindle protein in solid tumors, apoptosis inhibitor B-cell lymphoma (CLL / lymphoma) (BCL-2) in chronic myeloid leukemia; ribonucleotide reductase M2 (RRM2) in solid tumors; furin in solid tumors; polo-like kinase 1 (POL-1) in hepatocellular carcinoma 1) (PLK1), diacylglycerol acyltransferase 1 (DGAT1) in hepatitis C infection, beta-catenin in familial adenomatous polyposis; beta2-adrenergic receptor in glaucoma; RTP80l / Reddl (also known as DNA damage-inducible transcript 4 protein) in diabetic macular edema (DME) or age-related macular degeneration; vascular endothelial growth factor receptor I (VEGFR1) in age-related macular degeneration or choroidal neovascularization; caspase 2 in non-arteritic ischemic optic neuropathy; keratin 6AN17K mutant protein in pachyonychia congenita; influenza Influenza A virus genome / gene sequences in influenza infections; SARS coronavirus genome / gene sequences in severe acute respiratory syndrome (SARS) infections; respiratory syncytial virus genome / gene sequences in respiratory syncytial virus infections; Ebola virus genome / gene sequences in Ebola hemorrhagic fever infections; hepatitis B and C virus genome / gene sequences in hepatitis B and C infections; herpes simplex virus (HSV) genome / gene sequences in HSV infections, and coxsackievirus B3 genome / gene sequences in coxsackievirus B3 infections;Silencing pathogenic alleles of torsinA (TOR1A)-like genes (allele-specific silencing) in primary dystonia, pan-class I and HLA allele-specific in transplantation; and mutant rhodopsin gene (RHO) inhibitory nucleic acids in autosomal dominant retinitis pigmentosa (adRP); wherein the inhibitory nucleic acid is optionally an siRNA, antisense molecule, miRNA, RNAi, ribozyme, or shRNA;

[18] The method of any preceding claim, wherein the reaction product resulting from the activity of the enzyme extends the serum half-life of the gene therapy agent in the subject and / or extends or increases the subject's exposure to the gene therapy agent; Similar to the embodiments [1] to

[18] outlined above, the present invention also provides an enzyme described herein for use in any of the methods described in embodiments [1] to

[18] . The present invention further provides a gene therapy agent described herein for use in any of the methods described in embodiments [1] to

[18] . [Example]

[0108] Unless otherwise indicated, the methods used are standard biochemical and molecular biology techniques. Examples of suitable methodological textbooks include Sambrook et al., Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al., Current Protocols in Molecular Biology (1995), John Wiley and Sons Inc.

[0109] [Example 1] Optimal administration interval between immu- linidase and rATG Materials and Methods SCID mouse experimental setup and sample collection Immunodeficient CB17-SCID mice (Janvier Labs) were selected to evaluate the effects of immunoglobulin A (IgA) on gene transfer and AAV kinetics in vivo. SCID mice are unable to produce antibodies against common antigens, and precursor B and T cells fail to differentiate due to impaired VDJ rearrangement. However, SCID mice contain relatively normal numbers of nonlymphoid cells, such as NK cells, macrophages, and granulocytes. This SCID model was used to eliminate any mouse antibodies that, unlike human antibodies, are not efficiently cleaved by immunoglobulin A (IgA). To deliver human IgG to mice, an IgG antibody pool (IVIg, Privigen, Batch #P100322084) was used.

[0110] Female CB17-SCID mice (8 weeks old) were intraperitoneally (ip) administered 200 μL DPBS or untreated IVIg (80 mg / mL). One hour later, the mice were intravenously (iv) injected with 100 μL of 1 mg / mL immu- lidase or DPBS (Baxter, batch #0L008A). Four hours later, the mice were injected with an AAV viral vector (AAV8-CMV-LUC, 1x10 12 Mice were sacrificed on day 14, and a terminal blood sample was collected by cardiac puncture under isoflurane anesthesia. Tissue samples (heart and liver) were harvested, weighed, and flash-frozen in isopentane for subsequent AAV level analysis by qPCR.

[0111] Viral genome quantification At the end of the study, the gene transfer ability of the virus and its ability to reach target tissues were assessed by quantitative polymerase chain reaction (qPCR). The viral vector targeted the liver, which is also thought to be the organ where the virus is cleared from the circulation by Kupffer cells. Therefore, the results observed in the liver were further confirmed in the heart, which is used as another target tissue. The presence and clearance of virus in the blood after intravenous injection was monitored in plasma throughout the study and quantified by qPCR.

[0112] qPCR in plasma qPCR was used to quantify the number of AAV vector genomes in the circulation of mice at specific time points after AAV administration. Blood samples were collected into Li-Hep-coated vials at designated times (1, 4, 24, 48, 168, and 336 hours) after AAV administration and centrifuged at 2000 x g for 5 minutes at 4°C. Plasma was collected, transferred to 1.5 mL Eppendorf tubes, frozen on dry ice, and stored in a -80°C freezer until analysis.

[0113] Plasma samples from each mouse were diluted 1:20 with nuclease-free water in a 96-well plate and then incubated in a heat block at 70°C for 10 minutes to release viral DNA from the capsid. Samples were then analyzed by qPCR to detect virus-specific genes (ITRs). To each well of a 96-well plate, 10 μL of 2xS SoAdvanced Universal probe supermix (Biorad, Cat. #1725281, 1 μL TaqMan assay (primer sequences provided separately), 2 μL template, and 7 μL nuclease-free water were added. Each sample was measured in triplicate. The 96-well plate was sealed with an optical seal and run on an AriaMx Real-time PCR system under the following reaction conditions: 95°C for 5 minutes, followed by 45 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 15 seconds. The results were also confirmed by qPCR using additional primers targeting the luciferase gene (data not shown).

[0114] Using a standard curve (linearized AAV vector of known copy number / μL concentration), the Cq values ​​obtained by qPCR were converted to viral genome copies / μL plasma.

[0115] qPCR in liver lysates Quantitative PCR (qPCR) was applied to quantify the number of AAV vector genomes incorporated into the liver target tissue after viral administration.

[0116] At the end of the study (day 14), the entire liver was removed and weighed. One lobe was selected, isolated, and weighed for identification. It was then placed in a 2 mL Eppendorf tube containing 700 μL of 1x Cell Culture Lysis Reagent (Promega, Cat. #E1531) and 6.35 mm ceramic sphere beads (MP Biomedicals, Cat. #6540-412). The sample was homogenized using a Bead Mill 4 apparatus (Fisherbrand) at 5 m / s for three 30-second cycles with a 15-second pause between each cycle. The vial was then centrifuged at 20,000 x g for 2 minutes at 4°C. The resulting supernatant was transferred in two aliquots to 1.5 mL tubes and stored at -80°C until use. The remaining liver was divided into two parts, weighed, flash-frozen in isopentane, and stored at -80°C.

[0117] Total DNA (genomic, mitochondrial, and viral) was extracted from liver lysates using the DNeasy blood and tissue kit (Qiagen, Cat. #69504) according to the protocol provided by the manufacturer. Briefly, a portion of the lysate was used so as not to exceed the capacity of the DNA-binding column. The lysate was loaded onto a column containing a silica-based membrane that selectively binds DNA. After selective binding of DNA to the membrane, the bound DNA was washed and eluted with 10 mM Tris-HCl containing 0.5 mM EDTA, pH 9.

[0118] The extracted total DNA was diluted 25-fold with sterile water and then used as a template in qPCR reactions using two primer sets specific for either the viral genome or a genomic reference gene to allow calculation of the relative viral content per hepatocyte. Each well of a 96-well plate was added with 10 μL 2x SSoAdvanced Universal probe supermix (Biorad, Cat# 1725281), 1 μL TaqMan assay (20x, primer sequences provided separately), 5 μL DNA template, and 4 μL nuclease-free water. Each sample was run in triplicate, with triplicate wells containing primers detecting the viral-specific gene (ITR) and triplicate wells containing primers detecting the reference gene (actin). The 96-well plate was sealed with optical seals and run on an AriaMx Real-time PCR system under the following reaction conditions: 95°C for 3 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 30 seconds. The FAM signal was detected and ROX was used as a passive reference.

[0119] The Cq values ​​of the viral genes obtained were normalized to reference genes with constant abundance in the mouse genome. This allowed us to calculate the relative AAV genome content per cell, which could be used for comparison between groups. Samples without AAV treatment (injection of IVIg and immunofluorescence enzymes only) were used as calibrators to eliminate any background signal.

[0120] qPCR in the heart qPCR was performed to quantify the number of AAV vector genomes incorporated into cardiac target tissue after viral administration. At the end of the study (day 14), cardiac tissue samples were collected under isoflurane anesthesia, weighed, and flash-frozen in isopentane. Total DNA (genomic, mitochondrial, and viral) was extracted from cardiac tissue using the DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer's protocol. Briefly, a portion of cardiac tissue was excised, weighed, cut into small pieces, and lysed by incubation at 56°C for 4 hours in a buffer containing proteinase K. The resulting lysate was applied to a column with a silica-based membrane to selectively bind DNA, followed by washing and elution with 10 mM Tris-HCl containing 0.5 mM EDTA, pH 9.

[0121] The extracted total DNA was diluted 25-fold in sterile water and used as template in a qPCR reaction with two primer sets that bind to either a viral gene or a reference gene to allow calculation of the relative viral content per cardiac cell.

[0122] Each well of a 96-well plate was supplemented with 10 μL 2xSSoAdvanced Universal probe supermix (Biorad, Cat. #1725281), 1 μL TaqMan assay (20x, primer sequences provided separately), 5 μL DNA template, and 4 μL nuclease-free water. Each sample was run in triplicate. Amplification was performed using both primers detecting virus-specific genes (ITRs) and reference genes (actin or GAPDH) separately for each sample. The 96-well plate was sealed with optical seals and run on an AriaMx Real-time PCR system under the following reaction conditions: 95°C for 3 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 30 seconds. FAM signals were detected, and ROX was used as a passive reference. The resulting Cq values ​​of viral genes were normalized to reference genes present in a consistent amount in the mouse genome to calculate the relative AAV content per cell, which could be used for comparison between groups. A sample without AAV treatment (IVIg and immunofluorescence assay only) was used as a calibrator to eliminate any background signal. The primers used are shown in Table 1 below.

[0123] [Table 1]

[0124] result The results allowed for the comparison of AAV levels, as measured by qPCR, for up to 14 days after administration of the AAV gene therapy vector. The study included comparisons between three groups: · SCID mice administered human intravenous antibody (IVIg) followed by AAV; SCID mice administered human intravenous antibody (IVIg) and Ides followed by AAV; and SCID mice injected with AAV alone at the same time as the other mice The results obtained are shown in Figures 1 to 5. Figure 1 shows serum and plasma AAV levels for up to 7 days after AAV administration in the three groups. Figure 1 shows that in the group receiving IVIg and AAV (squares), AAV was rapidly cleared, with plasma AAV levels already near the limit of detection after 4 hours. In the group receiving AAV alone (circles), AAV levels declined more slowly and remained detectable even at day 7 (168 hours). In the group of mice receiving IVIg + Ides followed by AAV (triangles), AAV level kinetics were improved to a similar or even greater extent than AAV alone, with a delayed clearance rate and improved peak levels. Figure 2 shows the results of the same experiment as in Figure 1, but with serum and plasma AAV levels up to 48 hours after the start of the experiment. Again, Ides exerts beneficial effects even in the absence of IVIg administration. Figure 3 also shows the results of the same experiment, but now plots serum AAV levels as a bar graph at 1, 4, 24, and 48 hours after AAV administration, demonstrating that Ides administration counteracts the effect of IVIg and increases AAV levels at multiple time points measured, even in the absence of AAV. Figure 4 shows the AAV levels in the liver at the end of the experiment, 14 days after AAV administration. In the group administered AAV and IVIg, AAV was almost completely cleared by 14 days. In contrast, there was no significant difference in AAV levels in the liver after 14 days between the group administered AAV, IVIg, and Ides compared with the group administered AAV alone. Figure 5 shows the AAV levels in the heart after 14 days after AAV administration. Again, AAV was almost completely cleared in the group administered IVIg and AAV. The group administered AAV alone had significantly higher AAV levels in the heart after 14 days compared with the group administered IVIg, Ides, and AAV. However, the latter group still maintained much higher AAV levels compared with the group administered AAV and IVIg. Thus, the unexpected result was that the clearance rate of AAV from serum plasma in SCID mice was better than that in the group administered AAV alone without IVIg. Administration of IdeS also significantly delayed the clearance of AAV from the liver and heart on day 14, the final day of the experiment. Therefore, these results demonstrate that enzymes such as IdeS can be used to counteract the effects of antibodies on gene therapy and may be beneficial even in the absence of antibodies targeting the gene therapy vector.

Claims

1. 1. An enzyme for use in a method of treating a subject in need of gene therapy for a disease, said method of treating comprising the steps of: a. administering to the subject an enzyme in an amount effective to degrade or digest serum antibodies and / or inhibit or reduce effector functions of serum antibodies in the subject; and b. administering a gene therapy agent to the subject; wherein the gene therapy is administered at a dose lower than the minimum effective dose (MED) that would be administered to the subject in the absence of step a; The subject has either no detectable antibodies that specifically bind to the gene therapy agent, or a low titer of neutralizing antibodies (NAb) that specifically bind to the gene therapy agent, or a low titer of total antibodies (TAb) that specifically bind to the gene therapy agent, and optionally, the presence / titer of antibodies in the subject is measured in a serum sample obtained from the subject prior to step a.

2. The enzyme for use according to claim 1, wherein the subject has a titer of NAb of less than 1:10 or a titer of TAb of less than 1:

100.

3. 3. An enzyme for use according to claim 1 or 2, Step a is carried out in the range of about 4 hours to about 96 hours, in the range of about 4 hours to about 72 hours, in the range of about 4 hours to about 48 hours, preferably in the range of about 24 hours to 48 hours, preferably in the range of about 4 hours to about 24 hours, preferably in the range of about 4 hours to 6 hours, before step b; and / or Step a and / or step b are performed two or more times; and / or The enzyme, wherein the subject is a human.

4. 10. An enzyme for use according to any one of the preceding claims, comprising The dose of enzyme administered in step a is sufficient to degrade or digest all or substantially all IgG molecules present in the serum of said subject and / or inhibit or reduce the effector function of all or substantially all IgG molecules, and optionally the enzyme is an IgG cysteine ​​protease or an IgG endoglycosidase.

5. 5. An enzyme for use according to claim 4, (i) the IgG cysteine ​​protease is derived from a streptococcus such as Streptococcus pyogenes, Streptococcus equi or Streptococcus zooepidemicus, and optionally the enzyme is an IdeS, MAC2, SpeB, IdeZ or IdeE polypeptide, or (ii) The IgG endoglycosidase is derived from a streptococcus such as Streptococcus pyogenes, Streptococcus equi or Streptococcus zooepidemicus, or from Corynebacterium pseudotuberculosis, Enterococcus faecalis or Elizabethkingia meningoseptica, and optionally the enzyme is an EndoS, CP40, EndoE or EndoF2 polypeptide.

6. 7. An enzyme for use according to claim 5 or 6, the IgG cysteine ​​protease is a polypeptide comprising or consisting of a sequence that is at least 80% identical, such as at least 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 2, 4, 5, or the IgG cysteine ​​protease comprises or consists of the sequence of any one of SEQ ID NOs: 6 to 25 and 55 to 69, 91 or 92, optionally comprising an additional methionine at the N-terminus and / or a histidine tag at the C-terminus; or The IgG endoglycosidase is a polypeptide comprising or consisting of a sequence that is at least 80% identical, such as at least 85%, 90%, 95%, 99% or 100% identical to SEQ ID NO: 90, and optionally, the sequence comprises an additional methionine at the N-terminus and / or a histidine tag at the C-terminus.

7. 10. The enzyme for use according to any one of the preceding claims, wherein the enzyme is an immobilidase and / or EndoS.

8. The enzyme for use according to any one of the preceding claims, wherein the dose of said enzyme is in the range of 0.25 mg / kg to 0.5 mg / kg body weight, preferably in the range of 0.3 mg / kg to 0.5 mg / kg body weight.

9. 10. The enzyme for use according to any one of the preceding claims, wherein the gene therapy agent comprises a viral vector, optionally a lentiviral vector, an adenoviral vector or an adeno-associated viral (AAV) vector.

10. The gene therapy agent is 2×10 in the absence of step a. １１ ~2 x 10 １４ 2×10 １１ 10. The enzyme for use according to any one of the preceding claims, administered at a dose lower than

11. The viral vector is 2×10 １１ Vector genome (vg) / kg body weight (vg / kg) lower than 1 x 10 １０ vg / kg, less than 1 x 10 ９ vg / kg, less than 1 x 10 ８ vg / kg or less than 0.99 x 10 ７ 11. The enzyme for use according to claim 9 or 10, administered in a lower dose.

12. An enzyme for use according to any one of claims 9 to 11, The gene therapy comprises a lentiviral vector comprising an envelope protein to which the subject's antibody specifically binds; or The gene therapy comprises an AAV vector comprising a capsid protein to which the subject's antibody specifically binds, optionally comprising VP1, VP2 and / or VP3 capsid proteins.

13. 13. The enzyme for use according to claim 12, wherein the AAV vector comprises a VP1, VP2 and / or VP3 capsid protein having at least 60%, 70%, 80%, 90%, 95% or 100% sequence identity to a VP1, VP2 and / or VP3 capsid protein selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV3B, AAV-2i8, RhlO, Rh74, VP1, VP2 and / or VP3 capsid proteins of SEQ ID NO:93 and SEQ ID NO:

94.

14. 10. The enzyme for use according to any one of the preceding claims, wherein the disease is: (i) caused by a loss of protein function or activity, and the gene therapy agent comprises a heterologous polynucleotide encoding a protein or peptide that, when expressed in the subject, provides or complements the function or activity of the protein; or (ii) An enzyme that is caused by the gain of a function, activity or expression of a protein, and wherein the gene therapy agent comprises a heterologous polynucleotide that is transcribed into a nucleic acid that inhibits, reduces or decreases the expression of the function, activity or gain of expression of the protein.

15. 10. An enzyme for use according to any one of the preceding claims, comprising The disease is selected from the group consisting of: enzyme: proliferative diseases (cancer, tumor, dysplasia, etc.), metabolic diseases such as Crigler-Najjar syndrome and metabolic diseases of the liver, Friedreich's ataxia, infectious diseases, addiction (e.g., to tobacco, alcohol, or drugs), epilepsy, Canavan disease, adrenoleukodystrophy, viral diseases (including, e.g., hepatitis B or C virus, HIV, herpes, retroviruses, etc.), genetic diseases (muscle diseases such as cystic fibrosis, dystroglycanopathy, Duchenne muscular dystrophy, or dystrophies, myotubular myopathy, hemophilia A, hemophilia B, sickle cell anemia, sickle cell disease, Fanconi anemia, diabetes, amyotrophic lateral sclerosis (ALS), myotubulin myopathy, motor neuron diseases such as spinal muscular atrophy (SMA), spinal-bulbar muscular atrophy, or Charcot-Marie-Tooth disease, arthritis, severe combined immunodeficiency syndrome (RS-SCID, ADA-SCID, X-SCID, etc.), Wiskott-Aldrich syndrome, X-linked thrombocytopenia, X-linked congenital neutropenia, chronic granulomatous disease, etc.), coagulation factor deficiency, cardiovascular disease (restenosis, ischemia, dyslipidemia, homozygous familial hypercholesterolemia, etc.), eye diseases such as retinitis pigmentosa, Leber's congenital amaurosis, Leber's hereditary optic neuropathy, and Stargardt disease; lysosomal storage diseases such as Sanfilippo syndrome; hyperbilirubinaemia such as CN types 1 or 2 or Gilbert's syndrome; glycogen storage diseases such as Fabry disease, GSDI, GSDII (Pompe disease), GSDIII, GSDIV, GSDV, GSDVI, GSDVII, GSDVIII, and fatal congenital glycogen storage disease of the heart; and optionally an enzyme, wherein the vector comprises a therapeutic polynucleotide suitable for treating the disease.

16. 16. An enzyme for use according to claim 14(i) or claim 15, The subject has a disease characterized by the following: Pulmonary diseases (e.g., cystic fibrosis); bleeding disorders (e.g., hemophilia A or hemophilia B (with or without inhibitors)), thalassemia, blood disorders (e.g., anemia), Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), epilepsy, lysosomal storage diseases (e.g., aspartylglucosaminuria), Batten disease, late-onset infantile neuronal ceroid lipofuscinosis type II (CLN2), cystinosis, Fabry disease, Gaucher disease types I, II, and III, glycogen storage disease type II (Pompe disease), GM2-gangliosidosis type I (Tay-Sachs disease), GM2-gangliosidosis type II (Sandhoff disease), mucolipidosis type 1 (sialidosis types I and II), type II ( I-cell disease), Type III (pseudo-Hurler disease) and Type IV, mucopolysaccharidosis (Hurler disease and variants, Hunter, Sanfilippo types A, B, C, D, Morquio types A and B, Marote-Lamy and Sly disease), Niemann-Pick types A / B, C1 and C2, and Schindler types I and II), hereditary angioedema (HAE), copper or iron storage disorders (e.g., Wilson disease or Menkes disease), lysosomal acid lipase deficiency, neurological or neurodegenerative disorders, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, metabolic defects (e.g., glycogen storage diseases), solid organ diseases (e.g., brain, liver, kidney, heart), or viral infections (e.g., hepatitis B and C, HIV, etc.), bacterial or fungal diseases; a blood clotting disorder, optionally hemophilia A, hemophilia A with inhibitor antibodies, hemophilia B, hemophilia B with inhibitor antibodies, a deficiency of any of the following coagulation factors: factor VII, factor VIII, factor IX, factor X, factor XI, factor V, factor XII, factor II, von Willebrand factor, or combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase C1 deficiency, or gamma-carboxylase deficiency; Anemia, bleeding associated with trauma or injury, thrombosis, thrombocytopenia, stroke, coagulopathy, diffuse intravascular coagulation (DIC); excessive anticoagulant states associated with heparin, low molecular weight heparins, pentasaccharides, warfarin, small molecules antithrombotic (i.e., FXa inhibitors), or thrombocytopenia such as Bernard-Soulier syndrome, Glanzmann thrombasthenia, or storage pool deficiency.

17. 17. An enzyme for use according to any one of claims 1 to 13, 14(i), 15 or 16, wherein the heterologous polynucleotide encodes the enzyme: A protein selected from the group consisting of insulin, glucagon, growth hormone (GH), parathormone (PTH), growth hormone-releasing hormone (GRF), follicle-stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietin, angiostatin, granulocyte colony-stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), and tumor cells Growth factor alpha (TGFa), platelet-derived growth factor (PDGF), insulin-like growth factors I and II (IGF-I and IGF-II), TGF, activin, inhibin, bone morphogenetic proteins (BMPs), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neuturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog and tyrosine hydroxylase; or a protein selected from the group consisting of thrombopoietin (TPO), interleukins (IL-1 to IL-36), monocyte chemotactic proteins, leukemia inhibitory factor, granulocyte colony-stimulating factor, Fas ligand, tumor necrosis factors a and b, interferons a, b and g, stem cell factor, flk-2 / flt3 ligand, IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, MHC class I and class II molecules; or CFTR (cystic fibrosis membrane regulatory protein), a blood coagulation (clotting) factor) (factor XIII, factor IX, factor VIII, factor X, factor VII, factor Vila, protein C, etc.), gain-of-function blood coagulation factors, antibodies, retinal pigment epithelium-specific 65 kDa protein protein) (RPE65), erythropoietin, FDF receptor, lipoprotein lipase, ornithine transcarbamylase, b-globin, a-globin, spectrin, a-antitrypsin, adenosine deaminase (ADA), metal transporter (ATP7A or ATP7), sulfamidase, lysosomal storage disease-associated enzyme (ARSA), hypoxanthine guanine phosphoribosyltransferase, b-25 glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase, branched-chain ketoacid dehydrogenase, hormone, growth factor, insulin-like growth factor 1 or 2, platelet-derived growth factor, epidermal growth factor, nerve growth factor , neurotrophic factor-3 and -4, brain-derived neurotrophic factor, glial cell line-derived growth factor, transforming growth factor a and b, cytokines, a-interferon, b-interferon, interferon-g, interleukin-2, interleukin-4, interleukin-12, granulocyte-macrophage colony-stimulating factor, lymphotoxin, suicide gene products, herpes simplex virus thymidine kinase, cytosine deaminase, diphtheria toxin, cytochrome P450, deoxycytidine kinase, tumor necrosis factor, drug resistance proteins, tumor suppressor proteins (e.g., p53, Rb, Wt-1, NF1, von Hippel-Lindau (VHF)), adenomatous polyposis colicoli (APC)), peptides with immunomodulatory properties, tolerogenic or immunogenic peptides or proteins, Tregitope or hCDR1, insulin, glucokinase, guanylate cyclase 2D (FCA-GUCY2D), Rab escort protein 1 (Chroideremia), FCA5 (FCA-Febercillin), ornithine ketoacid aminotransferase (Gyrate Atrophy), Retinoschisin 1 (X-linked juvenile retinoschisis), USH1C (Usher's Syndrome 1C), X-linked recessive retinitis pigmentosa GTPase (X-linked retinitis pigmentosa GTPase (XFRP), MERTK (AR form of RP: retinitis pigmentosa), DFNB1 (Connexin 26 deficiency), ACHM2, 3 and 4 (Achromatopsia), PKD-1 or PKD-2 (Polycystic kidney disease), TPP1, CFN2, sulfotransferase, N-acetylglucosamine-L-phosphotransferase, cathepsin A, GM2-AP, NPC1, VPC2, sphingolipid activator protein, one or more zinc finger nucleases for genome editing and donor sequences used as one or more repair templates for genome editing; or A gene-editing nuclease, optionally a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALEN) or a functional Type II CRISPR-Cas9.

18. 16. An enzyme for use according to any one of claims 1 to 13, 14(ii) or 15, wherein the inhibitory nucleic acid binds to a gene, a transcript of said gene or a transcript of said gene associated with a polynucleotide repeat disease selected from the group consisting of: Huntington (HTT) gene, genes related to dentatorubral-pallidoluysian atrophy (atrophin1, ATN1), androgen receptor gene on the X chromosome in spinal-bulbar muscular atrophy, human Ataxin-1, -2, -3 and -7, Cav2. 1P / Q voltage-gated calcium channel (CACNA1A), TATA binding protein, Ataxin 8 reverse chain (ATXN80S), serine / threonine protein phosphatase 2A 55 kDa regulatory subunit B beta isoform in spinocerebellar degeneration (types 1, 2, 3, 6, 7, 8, 12, and 17), FMR1 (fragile X mental retardation 1) in fragile X syndrome, FMR1 (fragile X mental retardation 2) or AF4 / FMR2 family member 2 in fragile XE mental retardation; myotonin-protein kinase (MPK) in myotonic dystrophy; kinase (MT-PK); frataxin in Friedreich's ataxia; mutant superoxide dismutase 1 (SOD1) gene in amyotrophic lateral sclerosis; genes involved in the pathogenesis of Parkinson's disease and / or Alzheimer's disease; apolipoprotein B (APOB) and proprotein convertase subtilisin / kexin type 9 (PCSK9) in hypercholesterolemia; HIV Tat, human immunodeficiency virus transactivator response element of transcription genes in HIV infection; HIV TAR, HIV TAR, human immunodeficiency virus transcription activator human immunodeficiency virus transactivator response element gene; C-C chemokine receptor (CCR5) in HIV infection; Rous sarcoma virus (RSV) nucleocapsid protein in RSV infection, liver-specific microRNA (miR-122) in hepatitis C virus infection;p53 in acute renal failure, delayed graft function, or acute renal failure with renal impairment; protein kinase N3 (PKN3) in highly recurrent or metastatic solid malignant tumors; LMP2 (LMP2 is also known as proteasome beta subunit 9 (PSMB9)) in malignant melanoma; LMP7 (LMP7 is also known as proteasome beta subunit 9 (PSMB8)) in malignant melanoma; MECL1 (MECL1 is also known as proteasome beta subunit 10 (PSMB10)) in malignant melanoma; vascular endothelial growth factor (VEGF) in solid tumors; kinesin spindle protein in solid tumors, apoptosis inhibitor B-cell in chronic myeloid leukemia CLL / lymphoma (BCL-2); ribonucleotide reductase M2 (RRM2) in solid tumors; Furin in solid tumors; polo-like kinase 1 (PLK1) in hepatocarcinoma, diacylglycerol acyltransferase 1 (DGAT1) in hepatitis C infection, beta-catenin in familial adenomatous polyposis; beta-2 adrenergic receptor in glaucoma; RTP801 / Reddl (also known as DNA damage-inducible transcript 4 protein) in diabetic macular edema (DME) or age-related macular degeneration; vascular endothelial growth factor receptor I (VEGFR1) in age-related macular degeneration or choroidal neovascularization, caspase 2 in non-arteritic ischemic optic neuropathy; keratin 6AN17K mutant protein in congenital pachyonychia; influenza influenza A virus genome / gene sequence in influenza infection; SARS coronavirus genome / gene sequence in severe acute respiratory syndrome (SARS) infection; respiratory syncytial virus genome / gene sequence in respiratory syncytial virus infection; Ebola virus genome / gene sequence in Ebola hemorrhagic fever infection; hepatitis B and C virus genome / gene sequence in hepatitis B and C infection; herpes simplex virus (HSV) genome / gene sequence in HSV infection, coxsackievirus B3 genome / gene sequence in coxsackievirus B3 infection;Silencing of pathogenic alleles of torsinA (TOR1A)-like genes (allele-specific silencing) in primary dystonia, pan-class I and HLA allele-specific in transplantation; and mutant rhodopsin gene (RHO) inhibitory nucleic acids in autosomal dominant retinitis pigmentosa (adRP); wherein the inhibitory nucleic acid is optionally an siRNA, an antisense molecule, a miRNA, an RNAi, a ribozyme, or an shRNA enzyme;

19. An enzyme for use according to any preceding claim, wherein the reaction product resulting from the activity of the enzyme extends the serum half-life of the gene therapy agent in the subject and / or extends or increases the subject's exposure to the gene therapy agent.

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