Combination therapy for gene therapy

Abstract

The present invention relates to a method for enhancing gene therapy by using effective degradation or digestion of serum antibodies in a subject and / or inhibition or reduction of effector functions of serum antibodies in a subject. More specifically, the subject does not have a detectable antibody that specifically binds to gene therapy, or has a low titer neutralizing antibody (NAb) that specifically binds to gene therapy or a low titer total antibody (TAb) that specifically binds to gene therapy. The methods can increase the serum half-life of the therapy and indicate that lower gene therapy doses can be administered than other methods.

Description

Technical Field

[0001] The present invention relates to gene therapy, including compositions and methods for improving gene therapy, particularly in reducing the dose of the gene therapeutic agent required and / or prolonging the effect of gene therapy. Background Art

[0002] Gene therapy is a rapidly evolving and promising method, for example, for treating monogenic diseases. However, one problem encountered in gene therapy is that the body typically mounts an immune response to the gene therapeutic agent, particularly after administration of repeated doses of the gene therapeutic agent. Some gene therapy vectors are based on naturally occurring viruses, such as adeno-associated virus (AAV), where wild-type AAV infection is common, which explains why an individual may already have specific antibodies against the gene therapeutic agent prior to administration of gene therapy.

[0003] Antibodies against therapeutic agents, particularly gene therapeutic agents, are referred to as anti-drug antibodies (ADA). The production of ADA can reduce the transduction efficiency of gene therapy and promote more rapid clearance of the gene therapeutic agent. Thus, ADA can reduce the efficiency of the gene therapeutic agent. Some individuals may have a high titer of pre-existing ADA such that administration of the gene therapeutic agent as a treatment method is considered to be simply not suitable for them. In other individuals, after initial treatment with gene therapy, a high titer of ADA develops, and thus it is considered no longer safe to give them further doses of gene therapy, meaning that the initially effective treatment is no longer appropriate.

[0004] It is possible that ADA, whether pre-existing or developed after initial gene therapy treatment, represents an obstacle to the effectiveness of gene therapy for a given individual. But ADA may also mean there are safety issues, as high titers of the gene therapy vector are required to achieve the necessary effect. Given the history of gene therapy, where dose-related adverse events sometimes occur, the safety of any gene therapy is a critical factor, and thus any way to reduce the amount of gene therapy required is clearly desirable.

[0005] Therefore, given the impact of ADA, attempts to find ways to reduce the impact of ADA on gene therapy have been continuously focused on. Summary of the Invention

[0006] Surprisingly, it has been found that even if an individual has few or no serum antibodies against the gene therapeutic agent, administration of an enzyme capable of targeting and degrading serum antibodies prior to administration of gene therapy is still beneficial. Given the general belief that ADA is the cause of reduced gene therapy efficiency, this finding is unexpected in that administration of an enzyme that can target serum antibodies as well as the gene therapeutic agent is still beneficial even in the absence of or with low levels of specific antibodies against the gene therapeutic agent, given the focus on ADA in this field.

[0007] It has also unexpectedly been found that, for a general subject, the enzyme can be used to delay the clearance of gene therapy, particularly from the circulation, and thus increase the exposure of gene therapy in the specific subject. Accordingly, the present invention can also provide a method to reduce the dose of gene therapeutic agent to be administered to a subject, since the exposure of the gene therapeutic agent in the subject is increased.

[0008] Accordingly, the present invention provides a method for generally enhancing gene therapy and also helps to treat specific patients who were previously considered not to require administration of an enzyme during gene therapy. The cleavage product of the enzyme can have an unexpected inhibitory effect on the clearance of the gene therapy vector. By slowing down the clearance of the gene therapy vector and maintaining vector circulation, the opportunity to reach the target organ can be increased.

[0009] Accordingly, the benefits of the present invention can lie in reducing the amount of gene therapeutic agent administered, for example, reducing it to a lower dose than the dose usually administered. The benefit can be, or alternatively, increasing the half-life of the gene therapeutic agent in the body after administration. The benefit can be that a given amount of gene therapeutic agent can achieve a better effect. The benefit can be increasing the exposure of gene therapy in the subject. The benefit can be delaying the clearance time of gene therapy. The benefit can lie in increasing the amount of gene therapeutic agent reaching the target organ. The benefit can lie in being able to treat patient populations that were previously considered not to require administration of such enzymes. The benefit can lie in increasing gene transduction efficiency. Any of these benefits can be manifested individually or in combination with each other.

[0010] Accordingly, the present invention provides a method for treating a subject in need of gene therapy for a disease, comprising: a. administering to the subject an amount of an enzyme that is effective for degrading or digesting serum antibodies in the subject and / or inhibiting or reducing the effector function of serum antibodies in the subject; and b. administering gene therapy to the subject; wherein the administration dose of the gene therapy is lower than the minimum effective dose (MED) administered to the subject in the absence of step a; and wherein the subject does not have detectable antibodies that specifically bind to the gene therapy, or has a low titer of neutralizing antibodies (NAb) that specifically bind to the gene therapy or a low titer of total antibodies (TAb) that specifically bind to the gene therapy, optionally wherein 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 also provides a gene therapeutic agent for use in a method for treating a subject in need of gene therapy for a disease, the method comprising: a. Administering to the subject an amount of an enzyme that is effective to degrade or digest serum antibodies in the subject and / or inhibit or reduce the effector function of serum antibodies in the subject; and b. Administering gene therapy to the subject; wherein the dosage of the gene therapy is lower than the minimum effective dose (MED) administered to the subject in the absence of step a; and wherein the subject does not have detectable antibodies that specifically bind the gene therapy, or has a low titer of neutralizing antibodies (NAb) that specifically bind the gene therapy or a low titer of total antibodies (TAb) that specifically bind the gene therapy, optionally wherein 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 also provides an enzyme for use in a method of treating a subject in need of gene therapy for a disease, the method comprising: a. Administering to the subject an amount of an enzyme, wherein the enzyme is an amount effective to degrade or digest serum antibodies in the subject and / or inhibit or reduce the effector function of serum antibodies in the subject; and b. Administering gene therapy to the subject; wherein the gene therapy is administered at a dosage lower than the minimum effective dose (MED) administered to the subject in the absence of step a; and wherein the subject does not have detectable antibodies that specifically bind the gene therapy, or has a low titer of neutralizing antibodies (NAb) that specifically bind the gene therapy or a low titer of total antibodies (TAb) that specifically bind the gene therapy, optionally wherein 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 product resulting from the enzyme activity can prolong the serum half-life of the gene therapy in the subject and / or prolong or increase the exposure of the gene therapy in the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 . qPCR detection of AAV in the plasma of SCID mice up to 7 days after AAV injection. SCID mice were administered any one of the following: (a) AAV gene therapy alone (circles); (b) human IVIg followed by AAV gene therapy (squares); or (c) IVIg and IdeS followed by AAV gene therapy. After administering the AAV gene therapy, the AAV levels in the mouse serum were measured by qPCR for up to 7 days, as Figure 1 shown.

[0015] Figure 2. qPCR detection of AAV in the plasma of SCID mice up to 48 hours after AAV injection. Shown are the results of the same experiment as Figure 1 , but serum AAV levels are shown only up to 48 hours.

[0016] Figure 3 . qPCR detection of AAV in the plasma of SCID mice at 1 hour, 4 hours, 24 hours, and 48 hours after AAV injection. Shown are the results of the same experiment as Figure 1 and Figure 2 , but serum AAV levels are shown as bar graphs at the specified time points.

[0017] Figure 4 . qPCR detection of AAV in SCID hepatocytes 14 days after AAV injection. Shown are the results of the same experiment as Figures 1 to 3 , but AAV levels in hepatocytes are measured 14 days after AAV administration.

[0018] Figure 5 . qPCR detection of AAV in SCID heart cells 14 days after AAV injection. Shown are the results of the same experiment as Figures 1 to 4 , but AAV levels in heart cells are measured 14 days after AAV administration. Detailed implementation

[0019] 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.

[0020] 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.

[0021] 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.

[0022] SEQ ID NO: 4 is the mature sequence of IdeZ, lacking the N-terminal methionine and signal sequence.

[0023] SEQ ID NO: 5 is the sequence of the hybrid IdeS / Z. The N-terminus is based on IdeZ lacking the N-terminal methionine and signal sequence.

[0024] SEQ ID NO: 6 - 25 are the sequences of exemplary proteases used in the methods of the present invention.

[0025] SEQ ID NO: 26 is the sequence of the IdeS polypeptide. It contains the sequence of SEQ ID NO: 2, with an additional N-terminal methionine and histidine tag (internal reference pCART124).

[0026] SEQ ID NO: 27 is the sequence of the IdeZ polypeptide. It contains the sequence of SEQ ID NO: 4, with an additional N-terminal methionine and histidine tag (internal reference pCART144).

[0027] SEQ ID NO: 28 is the sequence of the IdeS / Z polypeptide. It contains the sequence of SEQ ID NO: 5, with an additional N-terminal methionine and histidine tag (internal reference pCART145).

[0028] SEQ ID NO: 29 is the contiguous sequence PLTPEQFRYNN, which corresponds to positions 63-73 of SEQ ID NO: 3.

[0029] SEQ ID NO: 30 is the contiguous sequence PPANFTQG, which corresponds to positions 58-65 of SEQ ID NO: 1.

[0030] SEQ ID NO: 31 is the contiguous sequence DDYQRNATEAYAKEVPHQIT, which corresponds to positions 35-54 of SEQ ID NO: 3.

[0031] SEQ ID NO: 32 is the contiguous sequence DSFSANQEIRYSEVTPYHVT, which corresponds to positions 30-49 of SEQ ID NO: 1.

[0032] SEQ ID NOs: 33-55 are the nucleotide sequences encoding the above proteases.

[0033] SEQ ID NOs: 56-69 are the sequences of exemplary proteases for use in the methods of the present invention.

[0034] SEQ ID NO: 70 is the contiguous sequence NQTN, which corresponds to positions 336-339 of SEQ ID NO: 1.

[0035] SEQ ID NO: 71 is the contiguous sequence DSFSANQEIR YSEVTPYHVT, which corresponds to positions 30-49 of SEQ ID NO: 1.

[0036] SEQ ID NOs: 72-86 are the nucleotide sequences encoding the polypeptides disclosed herein.

[0037] SEQ ID NO: 87 is the sequence SFSANQEIRY SEVTPYHVT, which corresponds to positions 31 - 49 of SEQ ID NO: 1.

[0038] SEQ ID NO: 88 is the sequence DYQRNATEAY AKEVPHQIT, which corresponds to positions 36 - 54 of the IdeZ polypeptide (NCBI Reference Sequence No. WP_014622780.1).

[0039] SEQ ID NO: 89 is the sequence DDYQRNATEA YAKEVPHQIT, which may be present at the N - terminus of the polypeptides of the present invention.

[0040] SEQ ID NO: 90 is the mature sequence of EndoS (endoglycosidase from Streptococcus pyogenes).

[0041] SEQ ID NO: 91 and SEQ ID NO: 92 are other exemplary proteases used in the methods of the present invention.

[0042] SEQ ID NO: 92 is the same as SEQ ID NO: 91, except that it lacks the first 20 residues consisting of the contiguous sequence DDYQRNATEAYAKEVPHQIT at the N - terminus of SEQ ID NO: 91.

[0043] SEQ ID NO: 93 is the amino acid sequence of the Spk1 protein.

[0044] SEQ ID NO: 94 is the amino acid sequence of the Spk2 protein.

[0045] Detailed Description of the Invention General Knowledge It should be understood that different applications of the disclosed products and methods can be adjusted according to the specific needs of the art. It should also be understood that the terms used herein are for the purpose of describing particular embodiments of the present invention only and are not intended to be limiting.

[0046] Furthermore, unless the context clearly dictates otherwise, the singular forms "a / an" and "the" as used in this specification and the appended claims include plural referents. Thus, for example, a reference to "polypeptide" includes "polypeptides" and the like.

[0047] "Polypeptide" is used herein in its broadest sense to refer to a compound having two or more subunit amino acids, amino acid analogs, or other peptidomimetics. Thus, the term "polypeptide" includes short peptide sequences as well as longer polypeptides and proteins. As used herein, the term "amino acid" refers to natural and / or unnatural or synthetic amino acids, including D or L optical isomers, as well as amino acid analogs and peptidomimetics.

[0048] The terms "patient" and "subject" are used interchangeably and generally refer to a human. Unless otherwise specified, references to IgG generally refer to human IgG.

[0049] When the term "comprising" is used herein, the invention also provides embodiments "consisting essentially of" and "consisting of" the recited content.

[0050] The amino acid identity as described above can be calculated using any suitable algorithm. For example, the PILEUP and BLAST algorithms can be used to calculate identity or to align sequences, such as to identify equivalent or corresponding sequences (usually in their default settings), e.g., as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S, F et al (1990)J Mol Biol 215:403-10. The software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. Such an algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy some positive-valued threshold score T when aligned with words of the same length in the 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 a search to find HSPs that contain them. The word hits are extended in both directions along each sequence as long as the cumulative alignment score can be increased. The extension of the word hits in each direction stops when: the cumulative alignment score drops from its maximum achieved value by the quantity X; the cumulative score becomes zero 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 the following as defaults: word length (W) of 11, BLOSUM62 scoring matrix (see Henikoffand Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) for alignments (B) of 50, expectation (E) of 10, M = 5, N = 4, and comparison of both strands.

[0051] The BLAST algorithm 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 gives an indication of the probability that a match occurs by chance between two nucleotide sequences or amino acid sequences. For example, in a comparison of a first sequence to a second sequence, if the minimum sum probability 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, the first sequence is considered similar to the second sequence. Alternatively, the UWGCG package provides the BESTFIT program, which can be used to calculate identity (e.g., using its default settings) (Devereux et al. (1984). Nucleic Acids Research 12, 387-395).

[0052] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0053] Enzymatic Treatment to Enhance Gene Therapy The present invention provides a method for improving the efficiency of gene therapy. For example, by using an enzyme capable of degrading or digesting serum antibodies in an object's body and / or inhibiting or reducing the effector function of serum antibodies in the object's body, the half-life of gene therapy can be extended. By using such an enzyme, the dose of the administered gene therapeutic agent can be less than the dose that would typically be administered in the absence of the enzyme. In certain cases, it may be possible to enhance gene therapy in an object using such an enzyme, even if the object has little or no such antibody. The exposure of gene therapy in the object's body can be increased and / or extended.

[0054] Accordingly, the present invention provides a method for treating an object in need of gene therapy for a disease, comprising: a. administering to the object an amount of an enzyme that is effective for degrading or digesting serum antibodies in the object's body and / or inhibiting or reducing the effector function of serum antibodies in the object's body; and b. administering gene therapy to the object; wherein the administered dose of the gene therapy is lower than the minimum effective dose (MED) that would be administered to the object in the absence of step a; and wherein the object does not have detectable antibodies that specifically bind to the gene therapy, or has a low titer of neutralizing antibodies (NAb) that specifically bind to the gene therapy or a low titer of total antibodies (TAb) that specifically bind to the gene therapy, optionally wherein the presence / titer of antibodies in the object's body is measured in a serum sample obtained from the object prior to step a.

[0055] Reaction products generated by enzyme activity prolong the serum half-life of gene therapy in a subject and / or increase or prolong the exposure of gene therapy in a subject.

[0056] When performing this method, steps a and b can each be performed only once. Steps a and b can also be performed more than once, such as two or more times. In one case, they are performed three, four, five or more times. The above method can be performed each time the subject needs gene therapy.

[0057] Steps a and b can be performed, for example, generally in the order described, so step a is performed first and then step b. In one case, steps a and b are performed at intervals of several hours to one month. They can be performed at intervals of several hours to two weeks. They can be performed at intervals of 4 hours to one week. In one case, they are performed at intervals of 4 hours to 48 hours. In a preferred case, they are performed at intervals of 4 hours to 24 hours. In a more preferred case, they are performed at intervals of 4 hours to 12 hours. However, in some cases, these two steps can be performed simultaneously. In one embodiment, both are performed simultaneously, separately or sequentially. Generally, both are provided as separate compositions. However, in one embodiment, the enzyme and the gene therapeutic agent are provided in the same composition. In another embodiment, the enzyme can also be administered after gene therapy, for example, for any of the above time periods, but after gene therapy is administered.

[0058] In one embodiment, after step a, a measurement can be performed to determine when to perform step b. For example, the level of specific antibodies against gene therapy can be measured. In one case, NAb can be measured. In another case, TAb can be measured. In one case, both can be measured. In another case, this measurement can be performed before step a to determine whether the subject is a subject without detectable antibodies specifically binding to gene therapy, or whether the subject is a subject with a low titer of NAb or a low titer of TAb, optionally where the presence / titer of antibodies in the subject is measured in a serum sample obtained from the subject before step a.

[0059] In some cases, measurements are performed using a biological sample from the subject. Preferred biological samples are blood or blood products. In certain embodiments, blood products include plasma or serum. A particularly preferred sample is serum.

[0060] The present invention also provides a method for enhancing the transduction efficiency of gene therapy treatment.

[0061] The present invention also provides a method for treating an object that does not exhibit pre-existing ADA or has only low ADA, where the ADA is specific for gene therapy, the method comprising: a. administering to the object an amount of the enzyme described herein; and b. administering to the object gene therapy.

[0062] The present invention also provides a method for enhancing the efficiency of gene therapy, the method comprising: a. administering to the object an amount of the enzyme described herein; and b. administering to the object gene therapy, wherein at least part of the enhanced efficiency is independent of the effect on any ADA present.

[0063] In one embodiment, the present invention is applied to objects who have only been given gene therapy before and not the enzyme because they do not exhibit an ADA response.

[0064] The present invention can be applied to any patient population mentioned herein. The present invention can also be used as a general method for enhancing gene therapy in patients. For example, the enzymes discussed herein can be used to extend the clearance time of gene therapy. The enzymes discussed herein can be used to increase the exposure time of gene therapy in the patient. The present invention can be used to increase the overall exposure of gene therapy in the patient, such as exposure level and / or exposure duration. The present invention can be used to increase the amount of gene therapy agent reaching a specific target organ. For example, the present invention can be used to increase the amount of gene therapy agent reaching the liver. The present invention can be used to increase the amount of gene therapy agent reaching the heart. The enzyme can be used to increase the amount of gene therapy agent reaching the muscle. The present invention can be used to extend the time that the gene therapy agent is present in such organs. The present invention can be used to increase the time that the gene therapy agent is present in the serum. The present invention can be used to increase the level of the gene therapy agent present in the serum. In one embodiment, an increase in the level of the gene therapy agent can be observed at least 48 hours after administration of the agent, such as at least 24 hours after administration.

[0065] It should be understood that the increase or extension is relative to a control method without the administration of the enzyme. This increase can be evaluated relative to a single control or the average of several experiments.

[0066] The present invention also provides a method for slowing the clearance of a gene therapy agent, wherein the method provides a. administering to the object an amount of an enzyme that is effective in degrading or digesting serum antibodies in the object's body and / or inhibiting or reducing the effector function of serum antibodies in the object's body; and b. administering to the object gene therapy.

[0067] For example, the gene therapeutic agent can be any of those described herein. For example, the enzyme can be any enzyme described herein. In one embodiment, the clearance rate can be slower overall, at least initially for 0 to 72 hours, compared to when the enzyme is not administered. In one embodiment, the clearance rate can be slower for at least 0 to 48 hours after administration of the gene therapeutic agent. In one embodiment, the clearance rate can be slower for at least 0 to 24 hours after administration of the gene therapeutic agent. In another embodiment, compared to when the enzyme is not administered, the overall exposure of the gene therapy in the subject can be increased for at least any of these time periods after administration of the gene therapeutic agent. In some embodiments, any time of administration of the enzyme and / or gene therapeutic agent can be used for any of the other embodiments described herein.

[0068] In another embodiment, the present invention provides the use of any antibody cleavage product of one of the enzymes described herein for slowing the rate of clearance of a gene therapeutic agent.

[0069] In a particularly preferred embodiment, the clearance rate or exposure rate is the clearance rate or exposure rate in the serum of the subject.

[0070] Enzyme The enzyme employed is an enzyme that is generally capable of degrading or digesting serum antibodies in the subject and / or inhibiting or reducing the effector functions of serum antibodies in the subject. The term "serum antibodies in the subject" can refer to any gamma immunoglobulin (IgG1, IgG2, IgG3, and IgG4) molecules present in human tissues or circulation prior to the performance of the method of the present invention.

[0071] The ability to degrade or digest serum antibodies and / or inhibit or reduce serum antibody effector functions can mean a reduction in the interaction of IgG molecules with Fc receptors. 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 the receptor family including FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIC (CD32C), FcγRIIIA (CD16a), and FcγRIIIB (CD16b). As used herein, the term FcγR includes the naturally occurring polymorphisms of FcγRI (CD64), FcγRIIA (CD32A), FcγRIIB (CD32B), FcγRIIC (CD32C), FcγRIIIA (CD16a), and FcγRIIIB (CD16b).

[0072] The enzyme used in the method of the present invention can be any enzyme that inactivates serum IgG, but is usually an IgG cysteine protease that cleaves IgG such that the antigen-binding domain and the Fc-interacting domain are separated from each other. In this case, the interaction of the Fc receptor of the serum IgG molecule is reduced because the number of intact IgG molecules in the serum is reduced. Alternatively, the enzyme can be an IgG endoglycosidase that cleaves the glycan structure on the Fc-interacting domain of IgG, particularly the N-linked biantennary glycan located at position Asn-297 (Kabat numbering). This glycan structure plays a key role in Fc receptor binding and complement activation. Thus, when it is removed in whole or in part from the protein, this will result in a decrease in Fc receptor binding or complement activation of the originally intact IgG molecule, and a decrease in recycling / half-life due to reduced binding to FcRn. The enzymes suitable for use will be discussed in more detail in the following section.

[0073] The enzyme is preferably administered by intravenous infusion. The enzyme can be administered by any suitable route, including, for example, intradermal, subcutaneous, transdermal, intramuscular, intraarterial, intraperitoneal, intraarticular, intraosseous, intranasal, or other suitable routes of administration. The enzyme can be administered more than once, for example, two, three, four, or more than four times. Preferably, the enzyme is administered twice.

[0074] The amount of enzyme administered can be from 0.01 mg / kg BW (BW - body weight) to 5 mg / kg BW, 0.01 mg / kg BW to 4 mg / kg BW, 0.01 mg / kg BW to 3 mg / kg BW, 0.01 mg / kg BW to 2 mg / kg BW, 0.01 mg / kg BW to 1 mg / kg BW, 0.01 mg / kg BW to 0.5 mg / kg BW, 0.01 to 0.3 mg / kg BW, preferably 0.1 mg / kg BW to 0.3 mg / kg BW, most preferably 0.2 mg / kg BW to 0.3 mg / kg BW. In one case, the dose of the enzyme is from 0.25 mg / kg BW to 0.5 mg / kg BW, preferably 0.3 mg / kg BW to 0.5 mg / kg BW. A particularly preferred dose is about 0.25 mg / kg BW. In one case, the dose of the enzyme is from 0.5 mg / kg BW to 1 mg / kg BW. A particularly preferred dose is about 1 mg / kg BW. In some embodiments, an enzyme of 2 × 1 mg / kg BW can be administered.

[0075] The amount of enzyme administered is typically sufficient to produce the desired effect, such as sufficient to extend the half-life of the gene therapy agent and / or reduce the amount of gene therapy agent required to produce the desired effect. The combination of the enzyme and gene therapy can produce a synergistic effect. Thus, the amounts of the enzyme and gene therapy can be sufficient to produce such a synergistic effect. For example, the effect can be a reduction in the amount of gene therapy agent that needs to be administered. Alternatively or additionally, it can be an extension of the half-life of the gene therapy agent. The half-life of the gene therapy agent can be measured by qPCR. A preferred method for measuring the serum half-life is to detect the gene therapy agent in the serum and / or plasma of the subject using qPCR.

[0076] In a particularly preferred case, the dose of the enzyme administered will be 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 functions of all or substantially all IgG molecules present in the serum of the subject, optionally wherein the enzyme is an IgG cysteine protease or an IgG endoglycosidase. Substantially all can mean at least 90%. Preferably, substantially all means at least 95%. More preferably, substantially all means at least 99%.

[0077] The enzyme can be administered to the same subject multiple times, provided that the amount of anti-drug antibody (ADA) in the subject's serum that can bind to the enzyme does not exceed a threshold determined by the clinician. Preferably, the enzyme is administered before the gene therapy agent. For example, the enzyme can be administered 4 hours to 96 hours before the administration of the gene therapy agent. For example, the enzyme can be administered 4 hours to 60 hours before the administration of the gene therapy. Preferably, the enzyme can be administered 24 hours to 60 hours before the administration of the gene therapy. More preferably, the enzyme can be administered 24 hours to 48 hours before the administration of the gene therapy agent. In one embodiment, the gene therapy is administered within 48 hours of the administration of the enzyme.

[0078] In some embodiments, the enzyme can be administered before and / or after the gene therapy. For example, the enzyme can be administered before the gene therapy as described above, and the enzyme can also be administered within a time period after the administration of the gene therapy. Thus, for example, the enzyme can also be administered within 48 hours after the gene therapy. The enzyme can be administered within 4 hours to 72 hours after the gene therapy. The enzyme can be administered within 4 hours to 6 hours after the gene therapy.

[0079] In some embodiments, the enzyme and gene therapy can be administered in a cycle. For example, each cycle can include administration at the times described above before the gene therapy, and optionally after the gene therapy. It can be repeated, for example, two, three, four, five or more times, and each repeated administration of the enzyme and gene therapy is considered a cycle.

[0080] In some embodiments, the present invention may include measuring ADA against a gene therapy agent, or such measurement has been performed on a subject. For example, an enzyme may be administered until the ADA level against the gene therapy agent is below a specific threshold.

[0081] The amount of ADA in the subject's serum that can bind to a protease can be determined by any suitable method, such as a reagent-specific CAP FEIA (ImmunoCAP) test or a titer assay. If the ADA in the subject exceeds the threshold, the treatment regimen may include administering a replacement enzyme. In one embodiment, the assay employed may be a luciferase-based assay. In another embodiment, the assay employed may be a bridging assay or a sandwich assay. In another embodiment, it may be an MSD assay.

[0082] Preferred enzymes include IgG cysteine proteases from Streptococcus bacteria, such as Streptococcus pyogenes, Streptococcus equi, Streptococcus equi subsp. zooepidemicus, or a newly described Streptococcus species temporarily designated as Streptococcus krösus Particularly preferred polypeptides include IdeS, MAC2, SpeB, IdeZ, IdeS / IdeZ hybrids (such as the enzymes described in WO2016 / 128558 or WO2021 / 233911), IgdE, Xork, or the IdE enzyme described in WO2021 / 254479.

[0083] Other preferred enzymes that can be employed include IgG endoglycosidases. Preferred IgG endoglycosidases are from Streptococcus bacteria, such as Streptococcus pyogenes, Streptococcus equi, or Streptococcus zooepidemicus, or from Corynebacterium pseudotuberculosis, Enterococcus faecalis, or Elizabethkingia meningoseptica. Particularly preferred enzymes are the EndoS, CP40, EndoE, or EndoF2 polypeptides. Particularly preferred is imlifidase. Further particularly preferred is EndoS.

[0084] IgG Cysteine Protease The IgG cysteine proteases used in the present invention are generally specific for IgG. In a preferred embodiment, the protease used in the method of the present invention is IdeS (immunoglobulin G-degrading enzyme of Streptococcus pyogenes), also known as imlifidase. IdeS is an extracellular cysteine protease produced by the human pathogen Streptococcus pyogenes. IdeS was initially isolated from a group A Streptococcus strain of serotype M1, but has now been identified in all tested group A Streptococcus strains IdesGene. IdeS has a very high substrate specificity, and the only substrate it can recognize is IgG. IdeS catalyzes a single proteolytic cleavage of the lower hinge region of the heavy chain of all subclasses of human IgG. IdeS also catalyzes the equivalent cleavage of the heavy chain of some IgG subclasses in various animals. IdeS effectively cleaves IgG into Fc and F(ab’)2 fragments through a two-stage mechanism. In the first stage, one (first) heavy chain of IgG is cleaved to produce a singly cleaved IgG (scIgG) molecule with a non-covalently bound Fc / 2 molecule. The scIgG molecule is actually an intermediate that retains the remaining (second) heavy chain of the original IgG molecule. In the second stage of this mechanism, this second heavy chain is cleaved by IdeS to release the F(ab')2 fragment and the homologous dimer Fc fragment. These are the products that are typically observed under physiological conditions. Under reducing conditions, the F(ab')2 fragment can dissociate into two Fab fragments, and the homologous dimer Fc can dissociate into its constituent monomers. IdeS has been shown to be particularly effective in cleaving human IgG. Within several hours after administration of IdeS, the entire plasma IgG pool is cleaved, and the IgG level in the blood remains low for about a week until newly synthesized IgG appears in the plasma. This indicates that the entire extracellular IgG pool rather than just the plasma pool (i.e., serum IgG molecules) is cleaved by IdeS (Winstedt et al.; PloS One 2015; 10(7): e0132011).

[0085] SEQ ID NO: 1 is the complete sequence of IdeS that includes an N-terminal methionine and a signal sequence. It is also available as NCBI reference sequence number WP_010922160.1. SEQ ID NO: 2 is the mature sequence of IdeS that lacks the N-terminal methionine and signal sequence. It is also available as Genbank accession number ADF13949.1.

[0086] In an alternative embodiment, the protease used in the method of the present invention is IdeZ, which is an IgG cysteine protease produced by Streptococcus equi subsp. zooepidemicus, a bacterium mainly found in horses. SEQ ID NO: 3 is the complete sequence of IdeZ that includes an N-terminal methionine and a signal sequence. It is also available as NCBI reference sequence number WP_014622780.1. SEQ ID NO: 4 is the mature sequence of IdeZ that lacks the N-terminal methionine and signal sequence.

[0087] In an alternative embodiment, the protease used in the method of the present invention is a hybrid IdeS / Z, such as the protease of SEQID NO: 5. The N-terminus is based on IdeZ that lacks the N-terminal methionine and signal sequence.

[0088] In a preferred embodiment, the protease for use in the method of the present invention may comprise SEQ ID NO:2, 4 or 5 or consist of SEQ ID NO: 2, 4 or 5. The protease for use in the present invention may comprise an additional N-terminal methionine (M) residue and / or a C-terminal tag to assist in expression and isolation from a standard bacterial expression system. 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). The histidine tag typically consists of six histidine residues, although it may be longer than this, typically up to 7, 8, 9, 10 or 20 amino acids, or shorter, such as 5, 4, 3, 2 or 1 amino acid.

[0089] In a further preferred embodiment, the protease for use in the present invention may comprise, consist essentially of or consist of any one of the sequences of SEQ ID NOs: 6 - 25. These sequences represent IdeS and IdeZ polypeptides with increased protease activity and / or reduced immunogenicity. Each of SEQ ID NOs: 6 - 25 may optionally comprise an additional N-terminal methionine and / or a C-terminal histidine tag. The histidine tag preferably consists of six histidine residues. The histidine tag is preferably linked to the C-terminus via a linker of 3× glycine or 5× glycine residues.

[0090] In a further preferred embodiment, the protease for use in the present invention may comprise, consist essentially of or consist of any one of the sequences of SEQ ID NOs: 56 - 69. These sequences represent IdeS polypeptides with increased protease activity and / or reduced immunogenicity. Each of SEQ ID NOs: 56 - 69 may optionally comprise an additional N-terminal methionine and / or a C-terminal histidine tag. The histidine tag preferably consists of six histidine residues. The histidine tag is preferably linked to the C-terminus via a linker of 3× glycine or 5× glycine residues.

[0091] In a further preferred embodiment, the protease for use in the present invention may comprise, consist essentially of or consist of: any one of the sequences of SEQ ID NOs: 6 - 25, optionally having up to 3 (such as 1, 2 or 3) amino acid substitutions. Each of SEQ ID NOs: 6 - 25 and its variants may optionally comprise an additional N-terminal methionine and / or a C-terminal histidine tag.

[0092] In a further preferred embodiment, the protease for use in the present invention may comprise, consist essentially of, or consist of: the sequence of any one of SEQ ID NOs: 56 - 69, optionally having up to 3 (e.g., 1, 2, or 3) amino acid substitutions. Each of SEQ ID NOs: 56 - 69 and its variants may optionally comprise an additional N-terminal methionine and / or a C-terminal histidine tag.

[0093] The length of the polypeptide of the present invention is generally at least 100, 150, 200, 250, 260, 270, 280, 290, 300, or 310 amino acids. The length of the polypeptide of the present invention generally does not exceed 400, 350, 340, 330, 320, or 315 amino acids. It should be understood that any of the lower limits listed above may be combined with any of the upper limits listed above to provide a length range for the polypeptide of the present invention. For example, the length of the polypeptide may be 100 to 400 amino acids, or 250 to 350 amino acids. The length of the polypeptide is preferably 290 to 320 amino acids, and most preferably 300 to 315 amino acids.

[0094] 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, particularly the amino acid sequences of SEQ ID NOs: 2, 4, or 5, respectively. The sequence of the protease of the present invention may comprise a variant of the amino acid sequence of SEQ ID NO: 2, 4, or 5, which has at least 80% identity with the amino acid sequence of SEQ ID NO: 2, 4, or 5. The variant sequence may have at least 80%, at least 85%, preferably at least 90%, at least 95%, at least 98%, or at least 99% identity with the sequence of SEQ ID NO: 2, 4, or 5. The variant may be the same as the sequence of SEQ ID NO: 2, 4, or 5, except for comprising one or more specific modifications specified in WO2016 / 128558 or WO2016 / 128559. The identity relative 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, or more preferably over the full length of SEQ ID NO: 4 or 5.

[0095] The proteases useful for use in the present invention can be IdeS, IdeZ or IdeS / Z polypeptides that are variants of the amino acid sequences comprising SEQ ID NO: 2, 4 or 5, wherein modifications are made relative to the sequences of SEQ ID NO: 2, 4 or 5, such as amino acid additions, deletions or substitutions. Such modifications are preferably conservative amino acid substitutions. Conservative substitutions replace an amino acid with another amino acid that is chemically similar, has similar chemical properties or has a similar side chain volume. The introduced amino acid can have a polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge similar to that of the amino acid it replaces. Alternatively, a conservative substitution can introduce another aromatic or aliphatic amino acid in place of a previously existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art.

[0096] IgG cysteine protease activity can be evaluated by any suitable method, such as by incubating a polypeptide with a sample containing IgG and determining the presence of IgG cleavage products. Suitable methods are described in WO2016 / 128559. Suitable assays include ELISA-based assays, such as those described in WO2016 / 128559. In such an assay, the wells of an assay plate are typically coated with an antibody target (e.g., bovine serum albumin (BSA)). Then a sample of the polypeptide to be tested is added to the wells, followed by a sample of a target-specific antibody, in this example a BSA-specific antibody. The polypeptide and the antibody are allowed to interact under conditions suitable for IgG cysteine protease activity. After a suitable time interval, 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 will bind to any intact target-specific antibody that has bound to the target in each well. After washing, the amount of detection antibody present in the well will be proportional to the amount of target-specific antibody bound to that well. The detection antibody can be conjugated directly or indirectly to a label or another reporting system (e.g., an enzyme), such that the amount of detection antibody remaining in each well can be determined. The higher the potency of the test polypeptide in the well, the less intact target-specific antibody remains, and thus the less detection antibody. Typically, at least one well on a given assay plate will contain IdeS instead of the polypeptide to be tested, such that the potency of the test polypeptide can be directly compared to the potency of IdeS. IdeZ and IdeS / Z can also be included for comparison. Another suitable assay is the MSD assay. In such an assay, the wells of an assay plate are typically coated with an antibody target (e.g., bovine serum albumin (BSA)). Then a sample of the polypeptide to be tested is added to the wells, followed by a sample of a target-specific antibody, in this example a BSA-specific antibody. The polypeptide and the antibody are allowed to interact under conditions suitable for IgG cysteine protease activity. After a suitable time interval, 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 will bind to any intact target-specific antibody that has bound to the target in each well. After washing, the amount of detection antibody present in the well will be proportional to the amount of target-specific antibody bound to that well. The detection antibody can be conjugated directly or indirectly to a SULFO tag that luminesces when a voltage in the instrument is applied to the plate electrodes.

[0097] Other assays can determine the potency of a test polypeptide by directly visualizing and / or quantifying IgG fragments generated by cleavage of IgG by the test polypeptide. Assays of this type are also described in WO2016 / 128559. Such assays typically incubate an IgG sample with a titration series of the test polypeptide at different concentrations (or with one or more of IdeS, IdeZ, and IdeS / Z as a control). The products generated by incubation at each concentration are then separated using gel electrophoresis, e.g., by SDS-PAGE. The intact IgG and the fragments generated by cleavage of IgG can then be identified by size and quantified by the staining intensity using a suitable dye. At a given concentration, the greater the amount of cleaved fragments, the greater the potency of the test polypeptide. The polypeptides of the present invention typically produce a detectable amount of cleaved fragments at a lower concentration (lower point in the titration series) than IdeZ and / or IdeS. Assays of this type can also identify whether the test polypeptide is more effective at cleaving the first or second heavy chain of the IgG molecule, since the amount of the different fragments generated by each cleavage event can also be determined. The polypeptides of the present invention can be more effective at cleaving the first chain of the IgG molecule than the second chain, particularly when the IgG is of the IgG2 isotype. The polypeptides of the present invention can be more effective at cleaving IgG1 than IgG2.

[0098] In a particularly preferred case, the IgG cysteine protease is a polypeptide comprising a sequence having at least 80% identity, such as at least 85%, 90%, 95%, 99% or 100% identity, with SEQ ID NO: 2, 4, 5, or a polypeptide consisting of said sequence. In a further particularly preferred case, the IgG cysteine protease comprises the sequence of any one of SEQ ID NO: 6-25 and 55-69, 91 or 92, or consists of said sequence. Optionally, the sequence comprises an additional methionine at the N-terminus and / or a histidine tag at the C-terminus.

[0099] IgG Endoglycosidase The enzyme can have the activity of an IgG endoglycosidase, preferably cleaving the glycan moiety at Asn-297 (Kabat number) in the Fc region of IgG. An example of such a protein is EndoS (endoglycosidase from Streptococcus pyogenes). EndoS hydrolyzes the β-1,4-di- N-N-acetylchitobiose core. The mature sequence of EndoS is provided as SEQ ID NO: 90. The agent can be a protein comprising or consisting of the amino acid sequence of SEQ ID NO: 90, or it can be a homolog thereof from an alternative bacterium such as Streptococcus equi or Streptococcus zooepidemicus, or Corynebacterium pseudotuberculosis, Enterococcus faecalis, or Elizabethkingia meningoseptica. The agent can be CP40, EndoE or EndoF2.

[0100] Alternatively, the agent can be a variant of the EndoS protein that comprises or consists of any amino acid sequence having at least 80%, 85%, 90% or 95% identity with SEQ ID NO: 90 and has IgG endoglycosidase activity. The variant of the EndoS protein can comprise or consist of: an amino acid sequence that has up to 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or more amino acid substitutions, insertions or deletions relative to the amino acid sequence of SEQ ID NO: 90, provided that the variant has IgG endoglycosidase activity. The amino acid substitutions are preferably conservative. Conservative substitutions are defined as previously.

[0101] Alternatively, the agent can be a protein that comprises or consists of a fragment of SEQ ID NO: 90 and has IgG endoglycosidase activity, preferably wherein the length of the fragment is 400 to 950, 500 to 950, 600 to 950, 700 to 950 or 800 to 950 amino acids. A preferred fragment consists of the first to the 409th amino acids of SEQ ID NO: 90, which corresponds to the enzymatically active EndoS α domain produced by cleavage with the streptococcal cysteine protease SpeB. The fragment can be produced by deleting one or more amino acid residues of 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 or more residues can be deleted. The deleted residues can be joined to other residues.

[0102] Any fragment or variant of SEQ ID NO: 90 preferably comprises residues 191 to 199 of SEQ ID NO: 90, namely Leu-191, Asp-192, Gly-193, Leu-194, Asp-195, Val-196, Asp-197, Val-198 and Glu-199 of SEQ ID NO: 90. These amino acids constitute the active site of the complete chitinase family 18, ending with glutamate. The glutamate in the chitinase active site is crucial for the enzyme's activity. Thus, most preferably, the variant of SEQ ID NO: 90 contains Glu-199 of SEQ ID NO: 90. The variant of SEQ ID NO: 90 may contain residues 191 to 199 of SEQ ID NO: 90 with one or more conservative substitutions, provided that the variant contains Glu-199 of SEQ ID NO: 90.

[0103] Gene Therapeutic Agent The gene therapy for use in the present invention can be any suitable gene therapy. It can be a gene therapeutic agent for treating any of the conditions mentioned herein. For example, the gene therapeutic agent can include a nucleic acid encoding a therapeutic gene of interest and means for its expression. It is possible that the therapeutic gene is capable of compensating for a non-functional or defective gene, such as a gene in any of the conditions mentioned herein. The gene therapy can be, for example, gene augmentation, gene silencing or suicide gene therapy. Preferably, it is gene augmentation therapy.

[0104] Preferably, the gene therapy is viral gene therapy. Examples of particularly preferred viral gene therapies are adeno-associated virus (AAV) gene therapy. Other examples of possible gene therapy vectors include adenoviral vectors. Other examples of gene therapy vectors include lentiviral and retroviral gene therapy vectors. Further examples of gene therapy vectors include CRISPR-based gene therapy vectors.

[0105] In one case, the gene therapeutic agent includes a lentiviral vector that contains an envelope protein that specifically binds to an antibody of the subject. In a preferred case, the gene therapy comprises an AAV vector that contains a capsid protein that specifically binds to an antibody of the subject. Optionally, the antibody in the subject specifically binds to the VP1, VP2 and / or VP3 capsid proteins.

[0106] Examples of AAV vectors that can be used include, for example, AAV serotype 1, AAV serotype 2, AAV serotype 3 (e.g., 3A and 3B), AAV serotype 4, AAV serotype 5, AAV serotype 6, AAV serotype 7, AAV serotype 8, AAV serotype 9, AAV serotype 10, AAV serotype 11, AAV serotype 12, AAV serotype 13, AAVrh32.33, AAVrh8, AAVrhlO, AAVrh74, AAVhu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAVAnc80, AAV PHP.B, and any other AAV now known or later discovered. See, e.g., BERNARD N. FIELDS et al., VIROLOGY, Volume 2, Chapter 69 (4th Edition, Lippincott-Raven Publishers). A variety of AAV serotypes and clades have been identified (see, e.g., Gao et al., (2004) J. Virology 78:6381-6388; Moris et al., (2004) Virology 33-:375-383). The AAV used can be a chimeric AAV, i.e., an AAV that contains a capsid protein having regions, domains, individual amino acids derived from two or more different AAV serotypes. The AAV vector can be developed by directed evolution.

[0107] In one embodiment, 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: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV3B, AAV-2i8, RhlO, Rh74, SEQ ID NO:93, and SEQ ID NO: 94, VP1, VP2, and / or VP3 capsid protein.

[0108] Gene therapy can be administered at a suitable dose. An advantage of the present invention is that the use of the enzyme generally results in a reduced amount of the gene therapy agent to be administered compared to the amount typically given in the absence of the enzyme. Thus, the enzyme and gene therapy show a synergistic combination. Examples of the reduction seen include a reduction of at least 5%. In one case, a reduction of at least 10%. In another case, a reduction of at least 20%. For example, in some cases, a reduction of at least 25%, 30%, 40%, at least 50% or more. This reduction is relative to the dose typically administered. In other examples, the reduction seen is within the range formed by any two of these values. In an alternative embodiment, instead of administering a reduced dose, the same dose is administered, but a better effect of the gene therapy is observed, for example, the expression level can be increased by at least any of the percentage values mentioned in this paragraph.

[0109] The amount of gene therapy typically given in the absence of the enzyme is known to those skilled in the art. For example, if the gene therapy has received marketing authorization, the amount will be listed in the package insert and / or the summary of product characteristics provided by the regulatory agency. Additionally or alternatively, the commonly used amounts are known in the art. For example, different AAV serotypes (AAV1, AAV2, AAV5, AAV8, and AAV9) have been used for gene therapy of these genetic disorders, with vector doses ranging from 2×10 11 to 2×10 14 vector genomes (vgs / kg) per patient (Maurya et al.; Cancer Gene Ther. 2022 Oct;29(10):1305 - 1306). Of course, the amount typically given will be evaluated relative to the same species, for example, when the subject is a human, then relative to a human.

[0110] In a preferred embodiment, the gene therapy agent is a viral vector, which is administered at a dose of less than 1×10 15 vector genomes (vg) / kg body weight (vg / kg). In some cases, the dose can be less than 10 14 vg / kg body weight. In some cases, the dose can be less than 10 13 vg / kg body weight. In some preferred cases, the dose can be less than 10 12 vg / kg body weight. In some more preferred cases, the dose can be less than 10 11 vg / kg body weight. In some even more preferred cases, the dose can be less than 1×10 10 vg / kg, less than 1×10 9 vg / kg, less than 1×10 8 vg / kg or less than 0.99×10 7vg / kg. The present invention can result in a reduction in the dosage to be administered to one of these levels. The dosage to be administered (but still effective) can be reduced to a level acceptable to subjects previously excluded from gene therapy.

[0111] When the gene therapy is a viral vector, it is typically administered at a dose of 2×10 11 to 2×10 14 . For example, the approved gene therapy vector therapies Elevidys™ and Zolgensma™ are administered at doses of 1.33×10 13 vg / mL and 1.1×10 14 vg / kg. The dosages typically administered for gene therapy (i.e., in the absence of step (a) of the present invention) are known to those skilled in the art, but can also be found in regulatory publications, such as the Summary of Product Characteristics of the drug. In the absence of step (a), gene therapy would be administered to a subject at a dose of 2×10 11 to 2×10 14 , while the present invention allows gene therapy to be administered at a dose lower than 2×10 11 (e.g., lower than 1×10 11 , 2×10 10 , 1×10 11 , 2×10 9 or 1×10 9 ). Thus, in one embodiment, gene therapy is administered at a dose lower than 2×10 11 (e.g., lower than 1×10 11 , 2×10 10 , 1×10 11 , 2×10 9 or 1×10 9 ), while in the absence of step (a), gene therapy would be administered to a subject at a dose of 2×10 11 to 2×10 14 .

[0112] In another embodiment, the gene therapeutic agent can be a non-viral vector. For example, the gene therapeutic agent can be selected from liposomes, nanoparticles, lipid nanoparticles, polymers, microparticles, microcapsules, micelles or extracellular vesicles.

[0113] The present invention can result in increased exposure of a gene therapy agent in a subject. It can be an increase in the duration of exposure. When the present invention is employed, the exposure at a given time point can be greater. The overall exposure can increase in terms of both exposure level and exposure duration. The present invention can result in an increased level of gene therapy reaching a given target organ. The overall exposure duration of the gene therapy agent can increase. The magnitude of any such increase can be, for example, at least 5%. It can be at least 10%. It can be at least 25%. It can be at least 50%. It can be at least 75%. It can be at least 100%. The increase seen can be a range with two of these values as endpoints.

[0114] The present invention provides for using the enzymes described herein to achieve any of the benefits specified herein, wherein a gene therapy is also administered to a subject.

[0115] Diseases and Conditions to be Treated Gene therapy can be used to treat almost any disease or disorder.

[0116] In a preferred case, the disease to be treated is caused by a loss of protein function or activity, and the gene therapy comprises a heterologous polynucleotide encoding a protein or peptide that, when expressed in the subject, provides or supplements the function or activity of the protein. In a further preferred case, the disease to be treated is caused by an increase in the functional activity or expression of a protein, and the gene therapy comprises a heterologous polynucleotide that transcribes into a nucleic acid that inhibits, reduces, or decreases the increase in the function, activity, or expression of the protein.

[0117] Preferred diseases and conditions that can be treated using the present invention include proliferative diseases (cancer, tumors, dysplasia, etc.), Crigler-Najjar syndrome, and metabolic diseases such as metabolic diseases of the liver, Friedreich's ataxia, infectious diseases, addictions (e.g., addiction to tobacco, alcohol, or drugs), epilepsy, Canavan disease, adrenoleukodystrophy, viral diseases (e.g., induced by hepatitis B or C virus, HIV, herpes, retroviruses, etc.), genetic diseases (cystic fibrosis, dystroglycanopathy, myopathies such as Duchenne muscular dystrophy or muscular dystrophy, myotubular myopathy, hemophilia A, hemophilia B, sickle cell anemia, sickle cell disease, Fanconi anemia, diabetes, amyotrophic lateral sclerosis (AFS), myotubularin myopathy, motor neuron diseases such as spinal muscular atrophy (SMA), spinal bulbar muscular atrophy or Charcot-Marie-Tooth disease, arthritis, severe combined immunodeficiency diseases (such as RS-SCID, ADA-SCID or X-SCID), Wiskott-Aldrich syndrome, X-linked thrombocytopenia, X-linked congenital neutropenia, chronic granulomatous disease, etc.), coagulation factor deficiencies, cardiovascular diseases (restenosis, ischemia, dyslipidemia, homozygous familial hypercholesterolemia, etc.), eye diseases such as retinitis pigmentosa, Leber congenital amaurosis, Leber hereditary optic neuropathy, and Stargardt disease; lysosomal storage diseases such as Sanfilippo syndrome; hyperbilirubinemia such as CN type I or II or Gilbert syndrome; Fabry disease, glycogen storage diseases such as GSDI, GSDII (Pompe disease), GSDIII, GSDIV, GSDV, GSDVI, GSDVII, GSDVIII, and lethal congenital cardiac glycogenosis.

[0118] In one embodiment, the subject to be treated has: - Pulmonary diseases (such as cystic fibrosis); hemorrhagic diseases (such as hemophilia A or hemophilia B with or without inhibitors), thalassemia, blood disorders (such as anemia), Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), epilepsy, lysosomal storage diseases (such as aspartylglucosaminuria, Batten disease, neuronal ceroid lipofuscinosis type 2 (CLN2) in late infancy, cystinosis, Fabry disease, Gaucher disease types I, II, and III, glycogen storage disease II (Pompe disease), GM2-gangliosidosis type I (Tay-Sachs disease), GM2-gangliosidosis type II (Sandhoff disease), mucolipidosis type I (sialidosis types I and II), mucolipidosis type II (I-cell disease), mucolipidosis type III (pseudo-Hurler disease), and mucolipidosis type IV, mucopolysaccharidoses (Hurler disease and variants, Hunter syndrome, Sanfilippo syndrome types A, B, C, D, Morquio syndrome types A and B, Maroteaux-Lamy syndrome, 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 accumulation disorders (such as Wilson's disease or Menkes disease), lysosomal acid lipase deficiency, neurological or neurodegenerative diseases, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, metabolic defects (such as glycogen storage diseases), diseases of solid organs (such as the brain, liver, kidney, heart), or infectious viral (such as hepatitis B and C, HIV, etc.), bacterial or fungal diseases; - Coagulation disorders, optionally hemophilia A, hemophilia A with inhibitory antibodies, hemophilia B, hemophilia B with inhibitory antibodies, deficiency of any coagulation factor among VII, VIII, IX, X, XI, V, XII, II, von Willebrand factor, or combined FV / FVIII deficiency, thalassemia, vitamin K epoxide reductase C1 deficiency, or gamma-carboxylase deficiency; - Anemia, bleeding related to trauma and injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); over-anticoagulation related to heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotic agents (i.e., FXa inhibitors), or platelet diseases such as Bernard-Soulier syndrome, Glanzmann thrombasthenia, or storage pool deficiency.

[0119] In another preferred embodiment, the gene therapy comprises an inhibitory nucleic acid that binds to a gene, a gene transcript, or a transcript of a gene associated with a polynucleotide repeat disease selected from the following: the huntingtin (HTT) gene, the gene associated with dentatorubral-pallidoluysian atrophy (ataxin 1, ATN1), the androgen receptor on the X chromosome in spinal and bulbar muscular atrophy, human ataxin-1, -2, -3, and -7, the Cav2.1 P / Q voltage-dependent calcium channel (CACNA1A), the TATA-binding protein, the ataxin 8 opposite strand (ATXN8OS), the serine / threonine-protein phosphatase 2A 55 kDa regulatory subunit B beta isoform in spinocerebellar ataxia (type 1, 2, 3, 6, 7, 8, 12, 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) in fragile XE mental retardation, or an AF4 / FMR2 family member 2; myotonic protein kinase (MT-PK) in myotonic dystrophy; frataxin in Friedreich's ataxia; the superoxide dismutase 1 (SOD1) gene mutant 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), hypercholesterolemia; HIV Tat, the human immunodeficiency virus transactivator protein of the transcription gene in HIV infection; HIV TAR, the human immunodeficiency virus transactivator response element gene in HIV infection; CC chemokine receptor (CCR5) in HIV infection; the Rous sarcoma virus (RSV) nucleocapsid protein in RSV infection, the liver-specific microRNA (miR-122) in hepatitis C virus infection; p53, acute kidney injury or delayed graft function or acute renal failure of kidney injury; protein kinase N3 (PKN3) in advanced recurrent or metastatic solid malignancies; LMP2, also known as proteasome subunit beta-9 type (PSMB9), metastatic melanoma; LMP7, also known as proteasome subunit beta-8 type (PSMB8), metastatic melanoma; MECL1, also known as proteasome subunit beta-10 type (PSMB10), metastatic melanoma; vascular endothelial growth factor (VEGF) in solid tumors; kinesin spindle protein in solid tumors, apoptosis inhibitor B-cell CLL / lymphoma (BCL-2) in chronic myeloid leukemia; ribonucleotide reductase M2 (RRM2) in solid tumors; furin in solid tumors; polo-like kinase 1 (PLK1) in liver tumors, diacylglycerol acyltransferase 1 (DGAT1) in hepatitis C infection, beta-catenin in familial adenomatous polyposis; beta2-adrenergic receptor, glaucoma;RTP801 / Redd1, 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 6A N17K mutant protein in congenital pachyonychia congenita; 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 infection; hepatitis B and hepatitis C virus genomes / gene sequences in hepatitis B and hepatitis C infections; HSV genome / gene sequence in herpes simplex virus (HSV) infection, Coxsackievirus B3 genome / gene sequence in Coxsackievirus B3 infection; such as torsin A (TOR1A) in primary dystonia, allelic silencing (allele-specific silencing) of the pathogenic alleles of genes of pan-class I and specific HLA alleles in transplantation; and mutant rhodopsin gene (RHO) in autosomal dominant retinitis pigmentosa (adRP); and wherein the inhibitory nucleic acid is optionally siRNA, antisense molecule, miRNA, RNAi, ribozyme or shRNA.;

[0120] The gene therapy agent can be a gene therapy agent that results in the expression of a missing or defective protein in a subject. For example, the present invention can be used to treat growth hormone deficiency. It can be used to treat immunodeficiency. In another case, the gene therapy agent can be a gene therapy agent that results in the killing of cells related to the underlying pathology of a disease state. More examples of diseases that can be treated include sickle cell anemia, SCID, CF, hemophilia, DMD, Huntington's disease, Parkinson's disease, hypercholesterolemia, alpha-trypsin deficiency, CGD, Fanconi anemia and Gaucher's disease.

[0121] Gene therapy is generally the treatment of directly administering a gene therapy agent to a subject. Treating the specified disorders or conditions herein includes preventing and treating the disorder. It can include alleviating or improving one or more symptoms of the disorder and even the overall condition.

[0122] The present invention also provides an enzyme for inactivating serum antibodies for use in a method for preventing or treating a disease or condition as described above. The present invention also provides the use of an enzyme for inactivating serum antibodies in a subject in the preparation of a medicament for preventing or treating a disease or condition in a method as described above.

[0123] The present invention also provides a gene therapy agent for use in a method of preventing or treating a disease or condition, wherein the method is as described above. The present invention also provides the use of the gene therapy agent in the preparation of a medicament for preventing or treating a disease or condition in the method as described above.

[0124] Subject to be Treated For the subject to be treated, they will typically have one of the conditions mentioned herein. Another advantage of the present invention is that it is effective even if the subject does not have antibodies against the gene therapy agent or only has low levels of such antibodies. Thus, in one case, the subject may not have detectable antibodies that specifically bind to the gene therapy. In another case, the subject may have a low titer of neutralizing antibodies (NAb) that specifically bind to the gene therapy. A neutralizing antibody can be, for example, an antibody that prevents the gene therapy agent from binding to its target or target cells. In one embodiment, the NAb titer of the subject can be <1:10, or the TAb titer can be <1:100. The titer of the NAb can be <1:50, <1:100, <1:500 or <1:1000. The titer of the TAb can be <1:500, <1:1000, <1:10,000 or lower.

[0125] Since the present invention enhances the effect of gene therapy, the subject can be a subject who was previously unable to be treated by gene therapy due to insufficient effect.

[0126] In other embodiments, the present invention can be applied to general patients, not just a specific group of patients. The present invention helps to improve gene therapy and can thus be applied to any patient who will benefit from gene therapy. For example, the present invention is capable of prolonging the exposure of the gene therapy agent in the subject, and this property can be used for general gene therapy patients, not just those with low or no serum antibodies.

[0127] The subject to be treated is typically a mammal. In a particularly preferred case, the subject to be treated is a human.

[0128] In one case, the subject can be a subject who has not previously received gene therapy. The subject can be a subject who has not previously received gene therapy as part of the method.

[0129] Compositions and Preparations Containing Polypeptides The present invention also provides compositions comprising the 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 therapeutic agents for use in the methods of the present invention. For example, the present invention provides compositions comprising a gene therapeutic agent and at least one pharmaceutically acceptable carrier or diluent.

[0130] The carrier needs to be "acceptable" in the sense of being compatible with the other ingredients of the composition and not injurious to the subject to whom the composition is administered. Usually, the carrier and the final composition are sterile and pyrogen-free.

[0131] The formulation of suitable compositions can be carried out using standard pharmaceutical formulation chemicals and methods, all of which are readily available to a reasonably skilled person. For example, the enzyme can be combined with one or more pharmaceutically acceptable excipients or solvents. Auxiliary substances such as wetting agents or emulsifying agents, pH buffering substances, reducing agents, etc. may be present in the excipient or solvent. Suitable reducing agents include cysteine, thioglycerol, thioredoxin, glutathione, etc. The excipients, solvents and auxiliary substances are generally pharmaceutical agents that do not induce an immune response in the individual receiving the composition and can be administered without causing 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 may also be included therein, such as inorganic acid salts, such as hydrochloride, hydrobromide, phosphate, sulfate, etc.; and organic acid salts, such as acetate, propionate, malonate, benzoate, etc. A detailed discussion of pharmaceutically acceptable excipients, solvents and auxiliary substances can be found in Remington’s Pharmaceutical Sciences; Mack Pub. Co., N.J. 1991.

[0132] Such compositions can be prepared, packaged, or sold in a form suitable for bolus administration or sustained delivery. Injectable compositions can be prepared, packaged, or sold in unit dosage forms (e.g., in an ampule with a preservative or in a multi-dose container). The compositions include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such compositions can also include one or more additional components, including 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 a dry (e.g., powder or granule) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) and then parenteral administration of the reconstituted composition. The composition can be prepared, packaged, or sold as a sterile injectable aqueous or oily suspension or solution. The suspension or solution can be formulated according to known techniques and can include additional components other than the active ingredient, such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable preparations can be prepared using a non-toxic parenterally acceptable diluent or solvent (e.g., water or 1,3-butanediol). Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils (e.g., synthetic mono- or di-glycerides of fatty acids).

[0133] Other available parenteral compositions include those that contain the active ingredient in microcrystalline form, in a liposomal formulation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation can include pharmaceutically acceptable polymers or hydrophobic materials, such as emulsions, ion exchange resins, slightly soluble polymers, or slightly soluble salts. The compositions can 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 infusion.

[0134] The compositions of the present invention are generally provided in an "effective amount" or "sufficient amount", which means an amount that, when administered as a single dose or multiple doses, alone or in combination with one or more other compositions, treatments, regimens or therapeutic agents, provides a detectable response, provides an expected or desired result or benefit to a subject for any duration (long-term or short-term), at any measurable or detectable level or for any duration (e.g., minutes, hours, days, months, years or longer). The dose of an "effective amount" or "sufficient amount" for treatment (e.g., to ameliorate or provide a therapeutic benefit or improvement) is generally effective in providing a response, to some measurable extent, to one, several or all of the adverse symptoms, consequences or complications of a disease, such as one or more adverse symptoms, conditions, pathologies or complications caused by or associated with the disease, although reducing, alleviating, inhibiting, suppressing, restricting or controlling the progression or exacerbation of the disease is also a satisfactory outcome.

[0135] Kit The present invention also provides a kit for practicing the methods described herein. The kits of the present invention may contain an enzyme or a composition comprising an enzyme as described above. The kit may include means for administering the enzyme or composition to a subject. The kit may further or alternatively contain a gene therapy agent or a composition comprising a gene therapy agent as described above. The kit may also include means for delivering the gene therapy agent. The kit may include instructions for the various components to be used in any of the methods described herein.

[0136] Further Preferred Embodiments The following embodiments represent further preferred embodiments, which do not currently form part of the claims, but may do so in the future.

[0137] [1] A method of treating a subject in need of gene therapy for a disease, comprising: a. administering to the subject an amount of an enzyme that is effective to degrade or digest serum antibodies in the subject's body and / or inhibit or reduce the effector function of serum antibodies in the subject's body; and b. administering gene therapy to the subject; wherein the dose of the gene therapy is lower than the minimum effective dose (MED) that would be administered to the subject in the absence of step a; and wherein the subject does not have detectable antibodies that specifically bind the gene therapy, or has a low titer of neutralizing antibodies (NAb) that specifically bind the gene therapy or a low titer of total antibodies (TAb) that specifically bind the gene therapy, optionally wherein the presence / titer of antibodies in the subject's body is measured in a serum sample obtained from the subject prior to step a; Optionally wherein the gene therapy is at a dose lower than 2×10 11administered at a dose of, whereas in the absence of step (a) the gene therapy would be administered to the subject at a dose of 2×10 11 to 2×10 14 .

[0138] [2] The method according to embodiment [1], wherein the subject has an NAb titer <1:10 or a TAb titer <1:100.

[0139] [3] The method according to embodiment [1] or [2], wherein: - step a is performed about 4 hours to about 48 hours, preferably about 24 hours to 48 hours, preferably about 4 hours to about 24 hours, preferably 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 subject is a human.

[0140] [4] The method according to any one of embodiments [1] to [3], 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 subject's serum and / or inhibit or reduce the effector functions of all or substantially all IgG molecules present in the subject's serum, optionally wherein the enzyme is an IgG cysteine protease or an IgG endoglycosidase.

[0141] [5] The method according to embodiment [4], wherein: (i) the IgG cysteine protease is from a bacterium of the genus Streptococcus, such as Streptococcus pyogenes, Streptococcus equi, or Streptococcus zooepidemicus, optionally wherein the enzyme is an IdeS, MAC2, SpeB, IdeZ, or IgdE polypeptide, or (ii) the IgG endoglycosidase is from a bacterium of the genus Streptococcus, such as Streptococcus pyogenes, Streptococcus equi, or Streptococcus zooepidemicus, or from Corynebacterium pseudotuberculosis, Enterococcus faecalis, or Elizabethkingia meningoseptica, optionally wherein the enzyme is an EndoS, CP40, EndoE, or EndoF2 polypeptide.

[0142] [6] The method according to embodiment [4] or [5], wherein: - the IgG cysteine protease is a polypeptide comprising a sequence having at least 80% identity, such as at least 85%, 90%, 95%, 99%, or 100% identity, with SEQ ID NO: 2, 4, 5, or consisting of said sequence, or wherein the IgG cysteine protease comprises the sequence of any one of SEQ ID NO: 6-25 and 55-69, 91, or 92, or consists of said sequence, optionally wherein the sequence comprises an additional methionine at the N-terminus and / or a histidine tag at the C-terminus; or - The IgG endoglycosidase is a polypeptide comprising a sequence having at least 80% identity, such as at least 85%, 90%, 95%, 99% or 100% identity, with SEQ ID NO: 90, or a polypeptide consisting of said sequence, optionally wherein said sequence comprises an additional methionine at the N-terminus and / or a histidine tag at the C-terminus.

[0143] [7] The method according to any one of embodiments [1] to [6], wherein the enzyme is imlifidase and / or EndoS.

[0144] [8] The method according to any one of embodiments [1] to [7], wherein the dose of the enzyme is from 0.25 mg / kg BW to 0.5 mg / kg BW, preferably from 0.3 mg / kg BW to 0.5 mg / kg BW.

[0145] [9] The method according to any one of embodiments [1] to [8], wherein the gene therapy comprises a viral vector, optionally a lentiviral vector, an adenoviral vector or an adeno-associated virus (AAV) vector.

[0146]

[10] The method according to embodiment [9], wherein the viral vector is at 2×10 11 vector genomes (vg) / kg body weight (vg / kg), less than 1×10 10 vg / kg, less than 1×10 9 vg / kg, less than 1×10 8 vg / kg or less than 0.99×10 7 and is administered at a dose.

[0147]

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

[10] , wherein: - the gene therapy comprises a lentiviral vector which comprises an envelope protein that specifically binds to the antibody of the subject; or - the gene therapy comprises an AAV vector which comprises a capsid protein that specifically binds to the antibody of the subject, optionally a VP1, VP2 and / or VP3 capsid protein.

[0148]

[12] The method according to embodiment

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

[0149]

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

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

[0150]

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

[13] , wherein the disease is selected from proliferative diseases (cancer, tumor, dysplasia, etc.), Crigler-Najjar syndrome, and metabolic diseases such as metabolic diseases of the liver, Friedreich's ataxia, infectious diseases, addiction (e.g., addiction to tobacco, alcohol, or drugs), epilepsy, Canavan disease, adrenoleukodystrophy, viral diseases (e.g., induced by hepatitis B or C virus, HIV, herpes, retrovirus, etc.), genetic diseases (cystic fibrosis, dystroglycanopathy, myopathies such as Duchenne myopathy or muscular dystrophy, myotubular myopathy, hemophilia A, hemophilia B, sickle cell anemia, sickle cell disease, Fanconi anemia, diabetes, amyotrophic lateral sclerosis (AFS), myotubularin myopathy, motor neuron diseases such as spinal muscular atrophy (SMA), spinal bulbar muscular atrophy, or Charcot-Marie-Tooth disease, arthritis, severe combined immunodeficiency diseases (such as RS-SCID, ADA-SCID, or X-SCID), Wiskott-Aldrich syndrome, X-linked thrombocytopenia, X-linked congenital neutropenia, chronic granulomatous disease, etc.), coagulation factor deficiencies, cardiovascular diseases (restenosis, ischemia, dyslipidemia, homozygous familial hypercholesterolemia, etc.), eye diseases such as retinitis pigmentosa, Leber congenital amaurosis, Leber hereditary optic neuropathy, and Stargardt disease; lysosomal storage diseases such as Sanfilippo syndrome; hyperbilirubinemia such as CN type I or II or Gilbert syndrome; Fabry disease, glycogen storage diseases such as GSDI, GSDII (Pompe disease), GSDIII, GSDIV, GSDV, GSDVI, GSDVII, GSDVIII, and lethal congenital cardiac glycogenosis; and optionally wherein the vector comprises a therapeutic polynucleotide suitable for treating the disease.

[0151]

[15] A method according to embodiment

[13] (i) or

[14] , wherein the subject has: - Pulmonary diseases (such as cystic fibrosis); hemorrhagic diseases (such as hemophilia A or hemophilia B with or without inhibitors), thalassemia, blood disorders (such as anemia), Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), epilepsy, lysosomal storage diseases (such as aspartylglucosaminuria, Batten disease, neuronal ceroid lipofuscinosis type 2 (CLN2) in late infancy, cystinosis, Fabry disease, Gaucher disease types I, II and III, glycogen storage disease II (Pompe disease), GM2-gangliosidosis type I (Tay-Sachs disease), GM2-gangliosidosis type II (Sandhoff disease), mucolipidosis type I (sialidosis types I and II), mucolipidosis type II (I-cell disease), mucolipidosis type III (pseudo-Hurler disease) and mucolipidosis type IV, mucopolysaccharidosis (Hurler disease and variants, Hunter syndrome, Sanfilippo syndrome types A, B, C, D, Morquio syndrome types A and B, Maroteaux-Lamy syndrome 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 accumulation disorders (such as Wilson's disease or Menkes disease), lysosomal acid lipase deficiency, neurological or neurodegenerative diseases, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, metabolic defects (such as glycogen storage diseases), diseases of solid organs (such as brain, liver, kidney, heart), or infectious viral (such as hepatitis B and C, HIV, etc.), bacterial or fungal diseases; - Coagulation disorders, optionally hemophilia A, hemophilia A with inhibitory antibodies, hemophilia B, hemophilia B with inhibitory antibodies, deficiency of any coagulation factor in VII, VIII, IX, X, XI, V, XII, II, von Willebrand factor, or FV / FVIII combined deficiency, thalassemia, vitamin K epoxide reductase C1 deficiency or gamma-carboxylase deficiency; - Anemia, bleeding related to trauma and injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); over-anticoagulation related to heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotic drugs (i.e., FXa inhibitors), or platelet diseases such as Bernard-Soulier syndrome, Glanzmann thrombasthenia or storage pool deficiency.

[0152]

[16] According to the method of any one of embodiments [1] to

[12] , embodiment

[13] (i),

[14] or

[15] , wherein the heterologous polynucleotide encodes: - A protein selected from insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (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), transforming growth factor a (TGFa), platelet-derived growth factor (PDGF), insulin-like growth factors I and II (IGF-I and IGF-II), TGF, activin, inhibin, bone morphogenetic protein (BMP), 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), neurocan, agrin, axonin-1 and axonin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog and tyrosine hydroxylase; or - A protein selected from thrombopoietin (TPO), interleukins (IL-1 to IL-36), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factor 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, class I and class II MHC molecules; or - CFTR (cystic fibrosis transmembrane conductance regulator), blood coagulation (clotting) factors (Factor XIII, Factor IX, Factor VIII, Factor X, Factor VII, Factor Vila, Protein C, etc.), increased functional coagulation factors, antibodies, retinal pigment epithelium-specific 65 kDa protein (RPE65), erythropoietin, FDF receptor, lipoprotein lipase, ornithine transcarbamylase, beta-globin, alpha-globin, spectrin, alpha-1 antitrypsin, adenosine deaminase (ADA), metal transporters (ATP7A or ATP7), sulfamidase, enzymes related to lysosomal storage diseases (ARSA), hypoxanthine-guanine phosphoribosyltransferase, beta-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, neurotrophin-3 and -4, brain-derived neurotrophic factor, glial cell line-derived growth factor, transforming growth factor alpha and beta, cytokines, alpha-interferon, beta-interferon, interferon-gamma, 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 (such as p53, Rb, Wt-1, NF1, von Hippel-Lindau (VHL), adenomatous polyposis coli (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 (achoroideremia), FCA 5 (FCA-Febercilin), ornithine ketoacid transaminase (gyrate atrophy), retinoschisin 1 (X-linked retinoschisis), USH1C (Usher syndrome 1C), X-linked retinitis pigmentosa GTPase (XFRP), MERTK (AR form of retinitis pigmentosa RP), DFNB1 (connexin 26 deafness), ACHM 2, 3 and 4 (achromatopsia), PKD-1 or PKD-2 (polycystic kidney disease), TPP1, CFN2, sulfatase, N-acetylglucosamine-1-phosphotransferase, cathepsin A, GM2-AP, NPC1, VPC2, sphingolipid activator proteins, one or more zinc finger nucleases for genome editing, and one or more donor sequences used as repair templates for genome editing; or - Gene editing nucleases, optionally including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or functional type II CRISPR-Cas9.

[0153]

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

[12] and embodiment

[13] (ii) or

[14] , wherein the inhibitory nucleic acid binds to a gene, a gene transcript, or a transcript of a gene associated with a polynucleotide repeat disease selected from: the huntingtin (HTT) gene, the gene associated with dentatorubral-pallidoluysian atrophy (atrophin 1, ATN1), the androgen receptor on the X chromosome in spinal and bulbar muscular atrophy, human ataxin-1, -2, -3, and -7, the Cav 2.l P / Q voltage-dependent calcium channel (CACNA1A), the TATA-binding protein, the ataxin 8 opposite strand (ATXN80S), the serine / threonine-protein phosphatase 2A 55 kDa regulatory subunit B beta isoform in spinocerebellar ataxia (type 1, 2, 3, 6, 7, 8, 12, 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) in fragile XE mental retardation, or an AF4 / FMR2 family member 2; myotonic protein kinase (MT-PK) in myotonic dystrophy; frataxin in Friedreich's ataxia; the superoxide dismutase 1 (SOD1) gene mutant 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), hypercholesterolemia; HIV Tat, the human immunodeficiency virus trans-activating protein of the transcription gene in HIV infection; HIV TAR, the human immunodeficiency virus trans-activating protein response element gene in HIV infection; the CC chemokine receptor (CCR5) in HIV infection; the Rous sarcoma virus (RSV) nucleocapsid protein in RSV infection; the liver-specific microRNA (miR-l22) in hepatitis C virus infection; p53, acute kidney injury or delayed graft function or acute renal failure in kidney injury; protein kinase N3 (PKN3) in advanced recurrent or metastatic solid malignancies; LMP2, also known as proteasome subunit beta-9 type (PSMB 9), metastatic melanoma; LMP7, also known as proteasome subunit beta-8 type (PSMB 8), metastatic melanoma; MECL1, also known as proteasome subunit beta-10 type (PSMB 10), metastatic melanoma; vascular endothelial growth factor (VEGF) in solid tumors; kinesin spindle protein in solid tumors, the apoptosis inhibitor B-cell CLL / lymphoma (BCL-2) in chronic myeloid leukemia; ribonucleotide reductase M2 (RRM2) in solid tumors; furin in solid tumors;Polo-like kinase 1 (PLK1) in liver tumors, diacylglycerol acyltransferase 1 (DGAT1) in hepatitis C infection, β-catenin in familial adenomatous polyposis; β2 adrenergic receptor, 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 6 A N17K 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 infection; hepatitis B and hepatitis C virus genomes / gene sequences in hepatitis B and hepatitis C infections; HSV genome / gene sequence in herpes simplex virus (HSV) infection, Coxsackievirus B3 genome / gene sequence in Coxsackievirus B3 infection; pathogenic allele silencing (allele-specific silencing) of genes such as torsin A (TOR1A) in primary dystonia, pan-class I and specific HLA alleles in transplantation; and mutant rhodopsin gene (RHO) in autosomal dominant retinitis pigmentosa (adRP); and wherein the inhibitory nucleic acid is optionally siRNA, antisense molecule, miRNA, RNAi, ribozyme or shRNA.;

[0154]

[18] The method according to any one of the preceding claims, wherein the reaction product generated by the activity of the enzyme extends the serum half-life of the gene therapy in the subject and / or extends or increases the exposure of the gene therapy in the subject.

[0155] In addition to the above embodiments [1] to

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

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

[18] . Examples

[0156] Unless otherwise stated, the methods used are standard biochemical and molecular biology techniques. Examples of suitable method 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.

[0157] Example 1 – Optimal Interval between Imlifidase and rATG Materials and Methods SCID Mouse Experiment Setup and Sample Collection Immunodeficient CB17-SCID mice (Janvier Labs) were selected to evaluate the effects of imlifidase on in vivo transduction and AAV kinetics. SCID mice are unable to produce antibodies against common antigens and their progenitor B and T cells cannot differentiate due to impaired VDJ rearrangement. However, SCID mice contain a relatively normal number of non-lymphocytes such as NK cells, macrophages, and granulocytes. The SCID model was used to exclude any contaminating murine antibodies that cannot be efficiently cleaved by imlifidase compared to human antibodies. Human IgG was introduced into these mice using IgG antibodies (IVIg, Privigen, lot # P100322084).

[0158] Female CB17-SCID mice (8 weeks old) were administered 200 µL of DPBS or intact IVIg (80 mg / mL) intraperitoneally (i.p.). One hour later, the mice were injected intravenously (i.v.) with 100 µl of imlifidase or DPBS (Baxter, lot #0L008A) at a concentration of 1 mg / mL. Four hours later, the mice were injected intravenously with 100 µl of AAV viral vector (AAV8-CMV-LUC, 1x10 12 vg / ml) or DPBS, so the AAV viral vector was administered 5 hours after the original IVIg injection. Throughout the study, blood samples were collected from the sublingual vein at specific time points relative to AAV injection. The mice were sacrificed on day 14 and terminal blood samples were collected by cardiac puncture under isoflurane anesthesia. Tissue samples (heart and liver) were dissected, weighed, and snap-frozen in isopentane for subsequent analysis of AAV levels by qPCR.

[0159] Quantification of Viral Genome At the end of the study, the transduction ability of the virus and thus its ability to reach the target tissue were evaluated by quantitative polymerase chain reaction (qPCR). The viral vector targets the liver, but the liver is also considered the organ where Kupffer cells clear the virus from the circulation. Therefore, another target tissue, the heart, was used to further confirm the results seen in the liver. Throughout the study, the presence and clearance of the virus in the blood after intravenous injection were tracked in plasma and quantified by qPCR.

[0160] Performing qPCR on Plasma To quantify the number of AAV vector genomes in the mouse blood circulation at given time points after administration, qPCR was performed. At predetermined time points after AAV administration (1 hour, 4 hours, 24 hours, 48 hours, 168 hours, 336 hours), blood was collected in Li-Hep-coated vials and centrifuged at 2000 x g for 5 minutes at 4°C. Plasma was extracted and transferred to 1.5 mL Eppendorf tubes, frozen on dry ice, and stored in an -80°C freezer until analysis.

[0161] Plasma samples from individual mice were diluted 1:20 in nuclease-free water in 96-well plates and then incubated in a heating block at 70°C for 10 minutes to release viral DNA from the capsid. Subsequently, the samples were analyzed by qPCR to detect the viral-specific gene (ITR). To each well of the 96-well plate, 10 µL of 2x SSoAdvanced Universal Probe Supermix (Biorad, Cat#1725281), 1 µL of TaqMan detection reagent (primer sequences provided separately), 2 µL of template, and 7 µL of nuclease-free water were added. Each sample was analyzed in triplicate. The 96-well plates were covered with an optical sealing film and run on an AriaMx real-time PCR system according to the following reaction conditions: 95°C for 5 minutes, then 45 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 15 seconds. The results obtained were further confirmed by qPCR using another primer targeting the luciferase gene (data not shown).

[0162] Using a standard curve (linearized AAV vector with known copy number / µL concentration), the Cq values obtained in qPCR were converted to viral genome copy number / µL plasma.

[0163] Performing qPCR on Liver Lysate To quantify the number of AAV vector genomes that successfully entered the liver target tissue after virus administration, quantitative PCR (qPCR) was performed.

[0164] At the end (day 14), the entire liver was dissected and weighed. A selected liver lobe was isolated, weighed for confirmation, and placed in a 2 mL Eppendorf tube containing 700 µL of 1x cell culture lysis reagent (Promega, Cat#E1531) and 6.35 mm ceramic beads (MP Biomedicals, Cat#6540-412). The sample was homogenized 3 times for 30 seconds at a speed of 5 m / s using a Bead Mill 4 device (Fisherbrand), with a 15-second pause between each time. The vial was centrifuged at 20,000 x g for 2 minutes at 4 °C, and the supernatant was transferred in two equal aliquots to 1.5 mL tubes and stored at -80 °C until use. The remaining liver was divided into 2 parts, weighed, quickly frozen in isopentane, and stored at -80 °C.

[0165] Total DNA (genomic, mitochondrial, and viral) was extracted from the liver lysate using the DNeasy Blood and Tissue Kit (Qiagen, Cat#69504) according to the manufacturer's protocol. Briefly, a portion of the lysate was used to avoid exceeding the capacity of the DNA-binding column. The lysate was applied to a column with a silica-based membrane that selectively binds DNA, and the bound DNA was subsequently washed and eluted with 10 mM Tris-HCl containing 0.5 mM EDTA, pH 9.

[0166] The extracted total DNA was diluted 25-fold in sterile water and then used as a template for the qPCR reaction. The qPCR reaction used two sets of primers specific for the viral genome or genomic reference genes to calculate the relative viral content per hepatocyte. To each well of a 96-well plate, 10 µl of 2x SSoAdvanced Universal Probe Supermix (Biorad, Cat#1725281), 1 µl of TaqMan detection reagent (20x, primer sequences provided separately), 5 µl of DNA template, and 4 µl of nuclease-free water were added. Each sample was analyzed in triplicate, with one set of three wells containing primers for detecting the virus-specific gene (ITR) and the other set containing the reference gene (actin). The 96-well plate was covered with an optical sealing film and run on an AriaMx real-time PCR system under the following reaction conditions: 95 °C for 3 minutes, then 40 cycles of 95 °C for 15 seconds and 60 °C for 30 seconds, detecting the FAM signal and using ROX as a passive reference.

[0167] The obtained Cq values of the viral gene were normalized relative to the reference gene, which is constantly present in the mouse genome, to generate the relative AAV genomic content / cell that can be used for comparison between groups. As a calibration to eliminate any background signal, samples untreated with AAV (only injected with IVIg and imlifidase) were used.

[0168] Performing qPCR on Heart Tissue To quantify the number of AAV vector genomes that successfully entered the cardiac target tissue after viral administration, qPCR was performed. At the end (day 14), cardiac tissue samples were collected under isoflurane anesthesia, weighed, and snap-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, and then minced, after which it was lysed at 56 °C for 4 hours in a buffer containing proteinase K. The lysate was applied to a column with a silica-based membrane that selectively binds DNA. Subsequently, the bound DNA was washed and eluted with 10 mM Tris-HCl containing 0.5 mM EDTA, pH 9. The extracted total DNA was diluted 25-fold in sterile water and then used as a template for the qPCR reaction, which used two sets of primers that bind to the viral or reference gene to calculate the relative viral content per cardiac cell.

[0169] To each well of a 96-well plate, 10 µl of 2x SSoAdvanced Universal Probe Supermix (Biorad, Cat#1725281), 1 µl of TaqMan detection reagent (20x, primer sequences provided separately), 5 µl of DNA template, and 4 µl of nuclease-free water were added. Each sample was analyzed in triplicate, and each sample was amplified separately using primers that detect the viral-specific gene (ITR) and the reference gene (actin or GAPDH). The 96-well plate was covered with an optical sealing film and run on an AriaMx real-time PCR system according to the following reaction conditions: 95 °C for 3 minutes, then 40 cycles of 95 °C for 15 seconds and 60 °C for 30 seconds, detecting the FAM signal and using ROX as a passive reference. The obtained Cq values of the viral gene were normalized to the reference gene, which is constantly present in the mouse genome, to generate the relative AAV content / cell that can be used for comparison between groups. As a calibration to eliminate any background signal, samples that were not treated with AAV (only injected with IVIg and imlifidase) were used. The primers used are given in Table 1 below.

[0170] Table 1: Primer sequences Results The results obtained by qPCR allowed comparison of AAV levels up to 14 days after administration of the AAV gene therapy vector. The experiment compared the following three groups: · SCID mice given human intravenous antibodies (IVIg) followed by AAV; · SCID mice that were given intravenous immunoglobulins (IVIg) and Ides and then AAV; and · SCID mice that were given AAV only at the same time points as other mice.

[0171] The results obtained are as Figures 1 to 5 shown.

[0172] Figure 1 shows the AAV levels in serum plasma up to 7 days after AAV administration in the three groups. Figure 1 shows that in the group given IVIg and AAV simultaneously (squares), AAV was cleared rapidly and the AAV level in plasma was close to the detection limit 4 hours later. In the group given AAV alone (circles), the AAV level decreased more slowly and was still detectable at day 7 (168 hours). In the group of mice injected with IVIg + Ides before AAV administration (triangles), the kinetics of the AAV level were similar to or even increased compared to those with AAV injection alone, with a slower clearance rate and increased peak levels. Figure 2 shows the results of the Figure 1 same experiment, but shows only the serum plasma AAV levels during the first 48 hours of the experiment. Again, the graph shows that Ides seems to confer a benefit even without the administration of IVIg. Figure 3 also shows the results of the same experiment, again the serum AAV levels, but this time shows the results at 1 hour, 4 hours, 24 hours, and 48 hours after AAV administration in the form of a bar graph. Again, the results show that the administration of Ides counteracts the effect of IVIg and seems to increase the AAV level at several measured time points even in the absence of IVIg.

[0173] Figure 4 shows the AAV levels in the liver at the end of the 14-day experiment after AAV administration. The group given AAV and IVIg had almost cleared AAV after 14 days. In contrast, there was no significant difference in the AAV level in the liver of the group given AAV, IVIg, and Ides compared to the group given AAV alone after 14 days. Figure 5 shows the AAV levels in the heart at 14 days after AAV administration. Again, the group given IVIG and AAV had almost completely cleared AAV. Although the AAV level in the heart of the group given AAV alone was indeed significantly higher at 14 days compared to the group given IVIg, Ides, and AAV, the AAV level in the group given IVIg, Ides, and AAV was still much higher compared to the group given IVIg plus AAV.

[0174] Thus, unexpectedly, when Ides were present, the clearance rate of AAV in the serum plasma of SCID mice was even lower than that of the group administered AAV without any IVIg. On day 14 at the end of the experiment, the administration of Ides also significantly slowed down the clearance of AAV from the liver and heart. Thus, the results suggest that enzymes such as Ides can be used to counteract the effects of antibodies on gene therapy and can be beneficial even in the absence of antibodies targeting gene therapy vectors.

Claims

1. An enzyme for use in a method of treating a subject in need of gene therapy for a disease, the method comprising: a. administering to the subject an amount of the enzyme effective to degrade or digest serum antibodies in the subject and / or inhibit or reduce the effector function of serum antibodies in the subject; and b. administering gene therapy to the subject; wherein the dosage of the gene therapy is lower than the minimum effective dose (MED) administered to the subject in the absence of step a; and wherein the subject does not have detectable antibodies that specifically bind the gene therapy, or has a low titer of neutralizing antibodies (NAb) that specifically bind the gene therapy or a low titer of total antibodies (TAb) that specifically bind the gene therapy, optionally wherein 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 according to claim 1, wherein the subject has an NAb titer < 1:10 or a TAb titer < 1:

100.

3. The enzyme according to claim 1 or 2, wherein: - step a is performed about 4 hours to about 96 hours, about 4 hours to about 72 hours, about 4 hours to about 48 hours, preferably about 24 hours to 48 hours, preferably about 4 hours to about 24 hours, preferably about 4 hours to 6 hours prior to step b; and / or - step a and / or step b is performed two or more times; and / or - the subject is a human.

4. The enzyme according to any one of the preceding claims, wherein the dosage 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 present in the serum of the subject, optionally wherein the enzyme is an IgG cysteine protease or an IgG endoglycosidase.

5. The enzyme according to claim 4, wherein: (i) the IgG cysteine protease is from a bacterium of the genus Streptococcus, such as Streptococcus pyogenes, Streptococcus equi, or Streptococcus zooepidemicus, optionally wherein the enzyme is an IdeS, MAC2, SpeB, IdeZ, or IgdE polypeptide, or (ii) the IgG endoglycosidase is from a bacterium of the genus Streptococcus, such as Streptococcus pyogenes, Streptococcus equi, or Streptococcus zooepidemicus, or from Corynebacterium pseudotuberculosis, Enterococcus faecalis, or Elizabethkingia meningoseptica, optionally wherein the enzyme is an EndoS, CP40, EndoE, or EndoF2 polypeptide.

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

7. The enzyme used according to any one of the preceding claims, wherein the enzyme is imlifidase and / or EndoS.

8. The enzyme used according to any one of the preceding claims, wherein the dose of the enzyme is from 0.25 mg / kg BW to 0.5 mg / kg BW, preferably from 0.3 mg / kg BW to 0.5 mg / kg BW.

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

10. The enzyme used according to any one of the preceding claims, wherein the gene therapy is administered at a dose lower than 2×10 11 whereas in the absence of step (a), the gene therapy would be administered to the subject at a dose of 2×10 11 to 2×10 14 .

11. The enzyme according to claim 9 or 10, wherein the viral vector is administered at a dose of less than 2×10 11 vector genomes (vg) / kg body weight (vg / kg), less than 1×10 10 vg / kg, less than 1×10 9 vg / kg, less than 1×10 8 vg / kg or less than 0.99×10 7 vg / kg.

12. The enzyme used according to any one of claims 9 to 11, wherein: - The gene therapy comprises a lentiviral vector, and the lentiviral vector comprises an envelope protein that specifically binds to the antibody of the subject; or - The gene therapy comprises an AAV vector, and the AAV vector comprises a capsid protein that specifically binds to the antibody of the subject, optionally a VP1, VP2 and / or VP3 capsid protein.

13. The enzyme used according to claim 12, wherein the AAV vector comprises a VP1, VP2 and / or VP3 capsid protein, and the VP1, VP2 and / or VP3 capsid protein has at least 60%, 70%, 80%, 90%, 95% or 100% sequence identity with a VP1, VP2 and / or VP3 capsid protein selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV3B, AAV-2i8, RhlO, Rh74, SEQ ID NO: 93 and SEQ ID NO: 94, VP1, VP2 and / or VP3 capsid protein.

14. The enzyme used 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 comprises a heterologous polynucleotide encoding a protein or peptide that provides or supplements the function or activity of the protein when expressed in the subject; or (ii) caused by an increase in the function, activity or expression of a protein, and the gene therapy comprises a heterologous polynucleotide transcribed into a nucleic acid that inhibits, reduces or decreases the expression of the increase in the function, activity or expression of the protein.

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

16. The enzyme according to claim 14(i) or 15, wherein the subject has: - Pulmonary diseases (such as cystic fibrosis); hemorrhagic diseases (such as hemophilia A or hemophilia B with or without inhibitors), thalassemia, blood disorders (such as anemia), Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), epilepsy, lysosomal storage diseases (such as aspartylglucosaminuria, Batten disease, neuronal ceroid lipofuscinosis type 2 in late infancy (CLN2), cystinosis, Fabry disease, Gaucher disease types I, II, and III, glycogen storage disease II (Pompe disease), GM2-gangliosidosis type I (Tay-Sachs disease), GM2-gangliosidosis type II (Sandhoff disease), mucolipidosis type I (sialidosis types I and II), mucolipidosis type II (I-cell disease), mucolipidosis type III (pseudo-Hurler disease), and mucolipidosis type IV, mucopolysaccharidoses (Hurler disease and variants, Hunter syndrome, Sanfilippo syndrome types A, B, C, D, Morquio syndrome types A and B, Maroteaux-Lamy syndrome, 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 accumulation disorders (such as Wilson's disease or Menkes disease), lysosomal acid lipase deficiency, neurological or neurodegenerative diseases, cancer, type 1 or type 2 diabetes, adenosine deaminase deficiency, metabolic defects (such as glycogen storage diseases), diseases of solid organs (such as the brain, liver, kidney, heart), or infectious viral (such as hepatitis B and hepatitis C, HIV, etc.), bacterial or fungal diseases; - Coagulation disorders, optionally hemophilia A, hemophilia A with inhibitory antibodies, hemophilia B, hemophilia B with inhibitory antibodies, deficiency of any coagulation factor among VII, VIII, IX, X, XI, V, XII, II, von Willebrand factor, or FV / FVIII combined deficiency, thalassemia, vitamin K epoxide reductase C1 deficiency or gamma-carboxylase deficiency; - Anemia, bleeding related to trauma and injury, thrombosis, thrombocytopenia, stroke, coagulopathy, disseminated intravascular coagulation (DIC); over-anticoagulation related to heparin, low molecular weight heparin, pentasaccharide, warfarin, small molecule antithrombotic drugs (i.e., FXa inhibitors), or platelet diseases such as Bernard-Soulier syndrome, Glanzmann thrombasthenia, or storage pool deficiency.

17. The enzyme as used in any one of claims 1 to 13, claim 14(i), 15, or 16, wherein the heterologous polynucleotide encodes: - a protein selected from insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (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), transforming growth factor a (TGFa), platelet-derived growth factor (PDGF), insulin-like growth factor I and II (IGF-I and IGF-II), TGF, activin, inhibin, bone morphogenetic protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin NT-3 and NT4 / 5, ciliary neurotrophic factor (CNTF), glial cell line-derived neurotrophic factor (GDNF), neurocan, agrin, axonin-1 and axonin-2, hepatocyte growth factor (HGF), ephrin, noggin, sonic hedgehog and tyrosine hydroxylase; or - a protein selected from thrombopoietin (TPO), interleukins (IL-1 to IL-36), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factor 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, class I and class II MHC molecules; or - CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), blood coagulation (clotting) factors (Factor XIII, Factor IX, Factor VIII, Factor X, Factor VII, Factor Vila, Protein C, etc.), increased functional coagulation factors, antibodies, retinal pigment epithelium-specific 65 kDa protein (RPE65), erythropoietin, FDF receptor, lipoprotein lipase, ornithine transcarbamylase, beta-globin, alpha-globin, spectrin, alpha-1 antitrypsin, adenosine deaminase (ADA), metal transporters (ATP7A or ATP7), sulfamidase, enzymes related to lysosomal storage diseases (ARSA), hypoxanthine-guanine phosphoribosyltransferase, beta-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, neurotrophin-3 and -4, brain-derived neurotrophic factor, glial cell line-derived growth factor, transforming growth factor alpha and beta, cytokines, alpha-interferon, beta-interferon, interferon-gamma, 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 (such as p53, Rb, Wt-1, NF1, von Hippel-Lindau (VHL), adenomatous polyposis coli (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 (achoroid), FCA 5 (FCA-Febercilin), ornithine ketoacid transaminase (gyrate atrophy), retinoschisin 1 (X-linked retinoschisis), USH1C (Usher syndrome 1C), X-linked retinitis pigmentosa GTPase (XFRP), MERTK (AR form of retinitis pigmentosa RP), DFNB1 (connexin 26 deafness), ACHM 2, 3 and 4 (achromatopsia), PKD-1 or PKD-2 (polycystic kidney disease), TPP1, CFN2, sulfatase, N-acetylglucosamine-1-phosphotransferase, cathepsin A, GM2-AP, NPC1, VPC2, sphingolipid activator proteins, one or more zinc finger nucleases for genome editing, and one or more donor sequences used as repair templates for genome editing; or - Gene editing nucleases, optionally including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or functional type II CRISPR-Cas9.

18. The enzyme used according to any one of claims 1 to 13 and claim 14(ii) or 15, wherein the inhibitory nucleic acid binds to a gene, a gene transcript, or a transcript of a gene associated with a polynucleotide repeat disease selected from the group consisting of: the huntingtin (HTT) gene, the gene associated with dentatorubral-pallidoluysian atrophy (atrophin 1, ATN1), the androgen receptor on the X chromosome in spinal bulbar muscular atrophy, human ataxin-1, -2, -3, and -7, the Cav2.lP / Q voltage-dependent calcium channel (CACNA1A), the TATA-binding protein, the ataxin 8 opposite strand (ATXN8OS), the serine / threonine-protein phosphatase 2A 55 kDa regulatory subunit B beta isoform in spinocerebellar ataxia (type 1, 2, 3, 6, 7, 8, 12, 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) in fragile XE mental retardation, or an AF4 / FMR2 family member 2; myotonic protein kinase (MT-PK) in myotonic dystrophy; frataxin in Friedreich's ataxia; the superoxide dismutase 1 (SOD1) gene mutant 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), hypercholesterolemia; HIV Tat, the human immunodeficiency virus transactivator protein of the transcriptional gene in HIV infection; HIV TAR, the human immunodeficiency virus transactivator protein response element gene in HIV infection; the CC chemokine receptor (CCR5) in HIV infection; the Rous sarcoma virus (RSV) nucleocapsid protein in RSV infection, the liver-specific microRNA (miR-122) in hepatitis C virus infection; p53, acute kidney injury or delayed graft function or acute renal failure in kidney injury; protein kinase N3 (PKN3) in advanced recurrent or metastatic solid malignancies; LMP2, also known as proteasome subunit beta-9 type (PSMB 9), metastatic melanoma; LMP7, also known as proteasome subunit beta-8 type (PSMB 8), metastatic melanoma; MECL1, also known as proteasome subunit beta-10 type (PSMB 10), metastatic melanoma; vascular endothelial growth factor (VEGF) in solid tumors; kinesin spindle protein in solid tumors, apoptosis inhibitor B-cell CLL / lymphoma (BCL-2) in chronic myeloid leukemia; ribonucleotide reductase M2 (RRM2) in solid tumors; furin in solid tumors; polo-like kinase 1 (PLK1) in liver tumors, diacylglycerol acyltransferase 1 (DGAT1) in hepatitis C infection, beta-catenin in familial adenomatous polyposis;β2 - adrenergic receptor, 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 6A N17K mutant protein in pachyonychia congenita; 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 infection; hepatitis B and hepatitis C virus genomes / gene sequences in hepatitis B and hepatitis C infections; HSV genome / gene sequence in herpes simplex virus (HSV) infection, Coxsackievirus B3 genome / gene sequence in Coxsackievirus B3 infection; pathogenic allele silencing (allele - specific silencing) of genes such as torsin A (TOR1A) in primary dystonia, pan - class I and specific HLA allele genes in transplantation; and mutant rhodopsin gene (RHO) in autosomal dominant retinitis pigmentosa (adRP); and wherein said inhibitory nucleic acid is optionally siRNA, antisense molecule, miRNA, RNAi, ribozyme or shRNA.; 19. The enzyme used according to any one of the preceding claims, wherein the reaction product generated by the activity of the enzyme prolongs the serum half-life of the gene therapy in the subject and / or prolongs or increases the exposure of the gene therapy in the subject.

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