Compositions and methods for binding and inhibiting neutralizing antibodies

Abstract

To provide a modified mycoplasma protein M.SOLUTION: Provided is a modified Mycoplasma protein M or a functional fragment thereof, wherein the modified Mycoplasma protein M or a functional fragment thereof has one or more amino acid mutations that increase or maintain the thermostability of the Mycoplasma protein M or a functional fragment thereof as compared to a wild-type Mycoplasma protein M or a functional fragment thereof.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] [Priority Statement] This application claims the benefit of U.S. Provisional Application No. 62 / 881,765, filed August 1, 2019, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present invention relates to methods and compositions for binding to antibodies. The methods can be used to isolate antibodies, treat disorders associated with excess antibodies, acutely block antibodies to stop autoimmune or inflammatory responses, and inhibit neutralizing antibodies. In one embodiment, the present invention relates to a method for inhibiting neutralizing antibodies against a heterologous agent when the heterologous agent is administered to a subject, comprising administering to the subject an effective amount of Mycoplasma protein M, or a functional fragment or derivative thereof, thereby inhibiting neutralization of the heterologous agent. The present invention further relates to modified Mycoplasma protein M, or a functional fragment thereof, having increased thermostability compared to wild-type protein M, and their use in the methods of the present invention. [Background technology]

[0003] Approximately one in ten people in the United States suffers from a rare genetic disease, which can have a significant impact on lifespan, quality of life, independence, and economic status. Gene therapy is the most promising form of treatment for correcting genetic diseases. Among gene therapies, the delivery vehicle, adeno-associated virus (AAV) vectors, has demonstrated therapeutic efficacy in numerous clinical trials. The first FDA-approved gene therapy was used in blind patients. The number of clinical trials involving AAV gene therapy for long-term therapeutic gene expression has increased substantially due to its safety and success in targeting many different types of organs. Despite clinical success, the main barrier to AAV-mediated gene delivery is the high incidence of neutralizing antibodies (NAb), which block vector transduction in target tissues. More than 90% of the general population has been exposed to AAV through natural infection, and more than 50% are seropositive for NAb to AAV. Because NAbs significantly attenuate therapeutic efficacy and cause variability in outcome, identification of pre-existing NAbs above a certain threshold during clinical trial screening renders patients ineligible for enrollment.

[0004] Several approaches have been employed to overcome AAV NAb resistance: serial plasma exchange, immunosuppressive drugs, and vector capsid modification to remove immune epitopes. Plasma exchange is inefficient, requiring multiple sessions to remove 2–3x the remaining NAb, and can only address low-titer NAb. Plasma exchange is also time-consuming, exposing patients to the risk of nosocomial infection transmission through concurrent antibody depletion and repeated intravenous needle exposure. Steroid or pharmacological immunosuppression to kill B cells poses significant health risks and requires long-term regimens to achieve a further 10-fold reduction in NAb resistance. AAV capsid engineering is an innovative approach, but ultimately insufficient against polyclonal anti-AAV sera, with only a modest 10-fold increase in antibody escape observed in vivo. Furthermore, modifying the capsid structure to alter NAb-recognition epitopes typically results in the production of less potent and incomplete vectors, as these altered surface regions are multifunctional. Current approaches do not successfully overcome pre-existing anti-AAV NAbs above the typical thresholds set for clinical trial exclusion or allow for repeated administration of the same AAV vector.

[0005] Mycoplasma protein M has been identified as being able to nonspecifically bind antibodies and block their ability to bind to antigens (U.S. Patent No. 9,593,150; U.S. Publication No. 2017 / 0320921). Summary of the Invention [Problem to be solved by the invention]

[0006] The present invention overcomes deficiencies in the art by providing a vector-independent, protein-based method for universally blocking Nabs, as well as other methods and compositions based on antibody binding. [Means for solving the problem]

[0007] The present invention is based, in part, on the development of a vector-independent, protein-based method for universally blocking NAbs and the demonstration that this method is effective across a wide range of existing NAb concentrations. The present invention exploits the use of a unique mycoplasma-derived protein and its analogs (termed Protein M) to enable successful gene delivery by preventing NAbs from neutralizing heterologous agents (e.g., AAV vectors) when administered to a subject. Protein M has been shown to block mammalian IgG, IgM, and IgA antibody classes in a species- and antigen-independent manner by universally binding to conserved regions on antibody light and heavy chains, resulting in structural interference with the antigen-recognition CDR regions. Protein M binds to antibodies with nanomolar affinity and prevents antigen-antibody binding for a variety of different immunoglobulin / antigen pairs tested. The inventors have found that Protein M blocks antibody recognition of AAV and prevents antibody-mediated neutralization of AAV. The level of AAV vector escape from NAb was shown to be proportional to NAb titer and volume, the amount of Protein M, and the amount of AAV. Protein M-mediated NAb escape was confirmed in vitro and in vivo using human serum (IVIG) and serum from AAV-immunized mice. It was shown that Protein M can be administered alone before AAV administration or formulated with AAV for NAb escape. The effectiveness of this approach depends on the interaction of Protein M with immunoglobulins before AAV neutralization. This approach can be used to overcome NAb to multiple heterogeneous agents (e.g., AAV vector serotypes) while maintaining the unique or beneficial properties of each agent for specific gene therapy applications.

[0008] The present invention further relates to the use of Protein M in other methods where binding to antibodies is beneficial, including the treatment of autoimmune disorders, the treatment of disorders associated with excess antibodies, methods of isolating antibodies, and methods of performing immunoassays.

[0009] The present invention further relates to modified protein M proteins that have increased thermostability and / or other advantageous characteristics for carrying out the methods of the invention in vivo or at elevated temperatures.

[0010] Thus, one aspect of the present invention relates to a modified Mycoplasma protein M or a functional fragment thereof having one or more amino acid mutations that increase or maintain the thermal stability of Mycoplasma protein M or a functional fragment thereof compared to wild-type Mycoplasma protein M or a functional fragment thereof.

[0011] A further aspect of the present invention relates to a polynucleotide encoding the modified mycoplasma protein M or a functional fragment thereof of the present invention, and a vector or transformed cell containing the same.

[0012] Another aspect of the present invention relates to a method for inhibiting neutralization of a heterologous agent by neutralizing antibodies when the heterologous agent is administered to a subject, comprising the step of administering to the subject an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, thereby inhibiting neutralization of the heterologous agent.

[0013] A further aspect of the present invention relates to a method for expressing a polypeptide or functional nucleic acid in a subject, comprising administering to the subject (a) a nucleic acid delivery vector encoding the polypeptide or functional nucleic acid, and (b) an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, thereby expressing the polypeptide or functional nucleic acid in the subject.

[0014] A further aspect of the present invention relates to a method for treating a disorder in a subject in need thereof, wherein the disorder is treatable by expressing a polypeptide or functional nucleic acid in the subject, comprising the steps of administering to the subject (a) a therapeutically effective amount of a nucleic acid delivery vector encoding the polypeptide or functional nucleic acid, and (b) an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, thereby treating the disorder in the subject.

[0015] Another aspect of the present invention relates to a method for editing a gene in a subject, comprising administering to the subject (a) a gene editing complex and (b) an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, thereby expressing a polypeptide or functional nucleic acid in the subject.

[0016] A further aspect of the present invention relates to a method for treating an autoimmune disease in a subject in need thereof, comprising administering to the subject an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, thereby treating the autoimmune disease.

[0017] A further aspect of the present invention relates to a method for treating a disorder associated with excess antibodies in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, thereby treating the disorder associated with excess antibodies.

[0018] Another aspect of the present invention relates to a method for isolating a compound comprising an antibody light chain variable region and / or heavy chain variable region from a sample, the method comprising the steps of contacting the compound with a modified Mycoplasma protein M or functional fragment thereof of the present invention attached to a solid support, and then eluting the compound from the modified Mycoplasma protein M or functional fragment thereof.

[0019] A further aspect of the present invention relates to a method for performing an immunoassay, the method comprising the step of using the modified Mycoplasma protein M of the present invention or a functional fragment thereof to bind to a compound comprising an antibody light chain variable region and / or a heavy chain variable region.

[0020] A further aspect of the present invention relates to a kit comprising the modified Mycoplasma protein M or a functional fragment thereof of the present invention.

[0021] These and other aspects of the present invention are set forth in greater detail in the description of the invention that follows. [Brief explanation of the drawings]

[0022] [Figure 1] We demonstrate that human IVIG contains AAV-neutralizing antibodies that inhibit AAV transduction in a dose-dependent manner. AAV2 neutralization plots were generated using a two-fold dilution series of human IVIG. Starting at 50 μg, IVIG was serially diluted down to less than 1 μg. Each dilution was incubated with 2 x 10 viral genomes of AAV2-luciferase at 4°C for 1 hour before seeding into individual wells. The luminescent reporter signal generated by AAV2 transgene expression is a functional readout of AAV transduction and is proportional to the amount of AAV available to transduce cultured HEK-293 cells. In this experiment, 1 x 10 cells were seeded into each well of a 48-well plate and transduced at an MOI of 2,000 in serum-free medium (n = 3 technical replicates per condition). Luciferase activity was measured 48 hours post-transduction on a plate reader after cell lysis and addition of luciferin. The relationship between the percentage of AAV neutralized for a given amount of IVIG was determined to be 72% (+ / - 1.3%) neutralization with 12.5 μg of IVIG compared to a no-IVIG control with an equivalent volume of phosphate-buffered saline (PBS). Furthermore, 25 μg of IVIG neutralized 96% (+ / - 0.2%) and 50 μg of IVIG neutralized 99% (+ / - 0.17%) of AAV2. [Figure 2]This shows that Protein M protects AAV from neutralizing antibodies present in human IVIG. Using 12.5 μg IVIG, which was previously shown to neutralize approximately 70% of AAV2 at a dose of 2 × 10⁻⁹ vg, antibody-mediated escape from neutralization was studied using a 2-fold dilution series of Protein M. In this case, we started with a molar ratio of 8 Protein M molecules to 1 IgG molecule, which represents a 4-fold excess of Protein M, considering that one IgG molecule has two Protein M binding sites that must be occupied for complete antibody inactivation. In each well, IVIG was first incubated with each Protein M dilution for 1 hour at 4°C, followed by the addition of AAV2-luciferase (2 × 10⁻⁹ vg) and an additional hour at 4°C. HEK-293 cells (1 × 10⁻⁹) were then added to the wells and incubated for 48 hours, after which the luciferase reporter signal was measured. Wells containing IVIG but no Protein M showed a reduction of approximately 68% (+ / - 3.3%) in AAV signal compared to phosphate-buffered saline (PBS)-containing no-IVIG control wells. However, Protein M dose-dependently blocked AAV neutralization, completely preventing neutralization (108%–193% of the no-IVIG control) at molar ratios higher than 2:1, whereas ratios of 1:1 or 0.5:1 only partially blocked neutralization (52% and 40% of the no-IVIG control, respectively). Furthermore, at ratios of 8:1 and 4:1, a dose-dependent increase in AAV luciferase signal was observed relative to the no-IVIG control. All wells were run in triplicate in serum-free medium in 48-well plates (n=3 technical replicates per condition), and luciferase signal was measured 48 hours after transduction. [Figure 3]These results demonstrate that excess Protein M provides little additional protection from NAbs at molar ratios higher than 8:1 and that Protein M enhances AAV transduction. In an attempt to establish the upper limit of Protein M molar ratios that protect or enhance AAV transduction, the NAb escape assay was repeated at 8:1 or 20:1 molar ratios, with or without neutralizing antibody conditions. Addition of 12.5 μg of IVIG neutralized the AAV2-luciferase signal by 80% (+ / - 2.6%) compared to PBS-without-IVIG control wells. Similar to previous experiments, preincubation of Protein M with 12.5 μg of IVIG (8:1 molar ratio, 33 μg Protein M) for 1 hour at 4°C, followed by incubation with AAV for 1 hour at 4°C, prevented AAV neutralization and enhanced the luciferase signal to 194% (+ / - 24%) of the PBS-without-IVIG control. At 10 times the amount of Protein M required for NAb escape (20:1 molar ratio, 75 μg Protein M), a modest increase in luciferase activity was observed to 256% (+ / - 44%) compared to the PBS-containing, no-IVIG control. In the presence of IVIG, the luciferase signal at the 20:1 ratio was not statistically different from that at the 8:1 ratio. Furthermore, without IVIG, when 75 μg or 33 μg Protein M alone was incubated with AAV2-luciferase for 1 hour at 4°C, there was no significant difference in the amount of luciferase signal (208% vs. 216%, respectively), confirming that the increase in luciferase signal compared to the PBS control was due to Protein M-based enhancement. Each well was seeded with 1 x 10 HEK-293 cells in serum-free medium and transduced at an MOI of 2,000 in a 48-well plate format (n = 3 technical replicates per well condition). [Figure 4]This shows that Protein M enhancement of AAV transduction is dose-dependent. Starting at 33 μg, a two-fold dilution series of Protein M demonstrates that incubation of Protein M with AAV2-luciferase results in an increase in luciferase signal compared to the PBS + AAV control. Luciferase signal enhancement disappears at concentrations of Protein M below 2 μg per 2 × 10 viral genomes, i.e., at a molar ratio of 40,000 molecules of Protein M per genome-containing AAV2-luciferase particle. Enhancement begins to saturate at approximately 33 μg of Protein M per 2 × 10 vector particles, i.e., approximately 700,000 molecules of Protein M per genome-containing AAV2-luciferase particle. Protein M dilutions were incubated with AAV at 4°C for 1 hour before transduction. Each well was seeded with 1 x 105 HEK-293 cells in serum-free medium and transduced at an MOI of 2,000 in a 48-well plate format (n = 3 technical replicates per well condition). [Figure 5]We demonstrate that protein M-mediated enhancement of AAV transduction depends on the direct interaction of protein M with the AAV capsid. Three different conditions were tested with AAV2-luciferase: preincubation of AAV with protein M 1 hour (-1 h) before transduction, addition of protein M to AAV at peri-transduction (0 h), or addition of protein M to wells 18 hours (18 h) after AAV transduction. Two negative control conditions without protein M were used: AAV alone was incubated in cell culture medium at 4°C for 1 hour before cell seeding and transduction (representing the conditions in the preincubation and peri-transduction groups), or AAV was diluted in PBS and added to the cell culture at the time of cell seeding and transduction (representing the conditions in the post-transduction group). Eighteen hours after transduction, an additional volume of PBS was added to the PBS control group to mimic the condition in which an equivalent volume of Protein M was added to the post-transduction group. No enhancement of AAV luciferase signal was observed in wells where Protein M was added near or after transduction, but a dose-dependent enhancement was observed in the pre-incubation group, indicating that the interaction between AAV and Protein M is necessary for the enhancement mechanism. All wells were seeded with 1 x 105 Huh7 cells, and each well was transduced with 2 x 108 vg of AAV, with n = 3 technical replicates per well condition in a 48-well plate format. [Figure 6]We demonstrate that Protein M does not prevent AAV neutralization after NAb binding to the capsid. AAV2-luciferase was first incubated with different dilutions of IVIG for 1 hour at 4°C, followed by the addition of Protein M and an additional hour at 4°C. A double negative control group received AAV alone, while a positive control group received 33 μg of Protein M incubated with AAV for 1 hour at 4°C before transduction. Three different dilutions of IVIG were used, including 200 μg, 50 μg, and 12.5 μg. Compared to the Protein M negative control group containing only AAV incubated with IVIG dilutions, 33 μg of Protein M did not allow post-neutralization enhancement or escape from neutralizing antibodies when 99% of AAV was already neutralized by 50 μg or 200 μg of IVIG. However, when 12.5 μg of IVIG was incubated with AAV, approximately 30% of the vector remained unneutralized (see Figures 1, 2, and 3), and 33 μg of Protein M was able to enhance transduction of this fraction after neutralization compared to Protein M negative control wells containing AAV and 12.5 μg of IVIG. Unlike previous figures, luciferase measurements were performed only 24 h after transduction, which may explain why the luciferase signal from the group with 12.5 μg of IVIG + AAV was less than 30% of that of the double negative control. Each well was seeded with 1 x 10 HEK-293 cells in serum-free medium and transduced at an MOI of 2,000 in a 48-well plate format (n = 3 technical replicates per well condition). [Figure 7]We demonstrate that preincubation of AAV with Protein M protects the vector from subsequent neutralization by IVIG. For this neutralization assay, the amount of Protein M was kept constant (8.25 μg), but various amounts of IVIG were used to achieve different molar ratios (50 μg to 3.12 μg of IVIG). Protein M was first incubated with AAV2-luciferase for 1 hour at 4°C, followed by the addition of IVIG and incubation at 4°C for 1 hour before transduction of cell cultures. Positive control groups contained AAV plus 33 μg, 8.25 μg, or 1 μg of Protein M, while negative control groups contained only AAV incubated with PBS. We observed a 2- to 3-fold enhancement of the non-neutralized fraction of AAV mediated by Protein M. Each well was seeded with 1 x 10 HEK-293 cells in serum-free medium and transduced at an MOI of 2,000 in a 48-well plate format (n = 3 technical replicates per well condition). Luciferase measurements were performed 24 hours after transduction. [Figure 8]We demonstrate that preincubation of AAV with Protein M protects vectors from IVIG-mediated neutralization in the presence of excess background serum immunoglobulins. For this experiment, we used 25 μg of IVIG, previously shown to neutralize approximately 95% of AAV2-luciferase at a dose of 2 × 10 viral genomes. 25 μg of human IVIG was added to cell culture wells containing 10% fetal bovine serum (FBS) (estimated bovine IgG concentration of 350 μg, determined by ELISA performed by the manufacturer). Another set of cell culture wells containing the same amount of 10% FBS served as a no-IVIG control. Protein M at molar ratios of 4:1, 2:1, and 1:1 (based on bovine IgG content) was incubated with AAV for 1 hour at 4°C, after which 10% FBS was added to the no-IVIG control wells or the wells containing 25 μg IVIG, respectively. In negative control groups, AAV was incubated with PBS instead of Protein M and added to wells containing 10% FBS or to wells containing 10% FBS with 25 μg of IVIG. The results show that preincubation with Protein M protected AAV2-luciferase from neutralization by IVIG, even at a ratio of one molecule of Protein M to one molecule of bovine IgG, although this amount of Protein M (108 μg) was still in a 12-fold greater ratio than human IVIG (25 μg). 1 × 10 HEK-293 cells were seeded in each well and transduced at an MOI of 2,000 in a 48-well plate format (n = 3 technical replicates per well condition). Luciferase measurements were performed 24 hours after transduction. [Figure 9]We demonstrate that Protein M enables in vitro escape of AAV8 from neutralizing antibodies found in pooled polyclonal serum collected from immunized mice. For this experiment, 10 μl of pooled polyclonal serum from AAV8-immunized mice was serially diluted in PBS, initially at a 10-fold dilution to generate wells containing 1 μl and 0.1 μl serum. The 0.1 μl serum wells were then further diluted in a 2-fold dilution series. AAV8-luciferase vector (2 × 10 viral genomes per well) was incubated with the neutralizing serum dilutions for 1 hour at 4°C prior to transduction. Protein M was added to the same 10-fold dilutions of AAV8-neutralized mouse serum at a ratio of approximately 2 molecules of Protein M to 1 molecule of immunoglobulin. Starting with 6.5 μg of Protein M to approximately 10 μg of immunoglobulin in 1 μl of neutralized mouse serum, each Protein M and serum sample was diluted independently and then combined for a 1-hour incubation at 4°C. All samples were then incubated with AAV8-luciferase at 4°C for 1 hour before being added to cells. Data from the resulting neutralization experiments were normalized to serum-free control wells containing AAV8 alone, and the luciferase signal was then fitted with a double exponential decay function to estimate a curve between the data points. Using the model curve, 50% neutralization of AAV8 by the polyclonal serum was found to occur at a volume of 0.0039 μl, corresponding to an effective titer of 1:2,564. However, 50% neutralization of the same serum incubated with a 2:1 molar ratio of Protein M occurred at a serum volume of 0.2744 μl, yielding an approximate effective titer of 1:36. This result demonstrates that Protein M can protect AAV over nearly a 100-fold difference in serum concentration in vitro. In a 96-well plate format, all wells were seeded with 5x104 HEK-293 cells (MOI of 4,000), n=3 technical replicates per well condition, and luciferase was measured 48 hours after transduction. [Figure 10]We demonstrate that in vivo administration of Protein M to mice passively immunized against AAV8 results in neutralizing antibody escape. In this experiment, mice were passively transferred via IV infusion with various volumes of polyclonal AAV8 serum (titer 1:2,564, as shown in previous figures) and then, within 15–20 minutes, IV administration of 2 × 10 viral genomes of AAV8-luciferase. This demonstrated a neutralization curve based on the serum volume delivered to each mouse. Compared to a group of naive mice not passively transferred with serum, 1 μl–0.001 μl of transferred serum resulted in greater than 50% neutralization of the AAV8-luciferase signal. However, when an estimated 2:1 ratio of Protein M (6.3 mg) was delivered to mice after passive transfer but before AAV administration, complete escape from neutralizing antibodies was achieved with 0.3 μl and 1 μl transfer serum volumes, indicating Protein M-mediated escape from neutralizing antibodies with a 1,000-fold change in NAb concentration. Furthermore, escape from neutralization was dose-dependent for the 0.3 μl passively transferred serum group, demonstrating reduced NAb escape for estimated molar ratios of 1:1 (3.15 mg) and 0.5:1 (1.58 mg) compared to the no-serum control group. For all groups, n = 5 mice per condition, and luciferase signal from the liver was measured 24 hours after AAV administration. [Figure 11] The average raw luminescence quantified from the results in FIG. 10 is shown. [Figure 12]This demonstrates that Protein M / antibody complexes are stable after in vitro formation. In this experiment, 1 μl of polyclonal serum (titer 1:2,564) containing an estimated 10 μg of immunoglobulin was incubated with Protein M (2:1 molar ratio, 6.6 μg) for various durations before the addition of AAV8-luciferase. Negative control groups contained neutralizing serum plus medium, while positive control groups contained medium plus PBS or Protein M plus medium. Incubation intervals of 72, 48, 24, 16, 4, 2, and 1 hour were applied to all groups. All incubation durations demonstrated Protein M-mediated protection of AAV8 from neutralization compared to the negative control of medium plus PBS. In a 96-well plate format, 5 x 10 HEK-293 cells were transduced at an MOI of 4,000 with AAV8-luciferase added to the wells at the time of seeding (0 h) (n = 3 technical replicates per well condition). Luciferase measurements were performed 48 h post-transduction. [Figure 13] This shows that the efficacy of Protein M-mediated neutralizing antibody escape in vivo is unstable. The same experiments as in Figures 10 and 11 were performed, but this time, AAV8 was added 5 minutes after Protein M administration or 3 hours after Protein M administration to test the durability of NAb escape. The AAV8-luciferase signal was approximately one-third of that of the no-serum control group after waiting 3 hours before AAV8 administration, but when AAV8 administration was waited only 5 minutes after Protein M, the luciferase signal was nearly equivalent to that of the no-serum control group. An approximate 2:1 ratio of Protein M (6.3 mg) was delivered to mice after passive transfer but before AAV administration, with n = 5 mice per condition per group, except for the 3-hour interval group, which contained n = 3 mice. Luciferase signals from the liver were measured 24 hours after AAV administration. [Figure 14]We demonstrate that truncated protein M from Mycobacterium genitalium (MGWT) is unstable at body temperature. The melting temperature (Tm) was determined using nanodifferential scanning fluorometry (NanoDSF). The inflection point of the first derivative indicates the Tm. The protein was recombinantly expressed in E. coli and purified using nickel column chromatography prior to measurement. Circular dichroism assays demonstrated that MGWT was stable at 20°C for at least 2 hours without the formation of visible precipitates. However, at 37°C, MGWT began to unfold after 15 minutes, forming visible precipitates. This indicates that the protein is unstable near standard human body temperature. A temperature ramp from 0 to 100°C demonstrated immediate unfolding of MGWT and a melting temperature (Tm) of approximately 41.2°C. Unfolding was accompanied by aggregation of visible precipitates. [Figures 15A-15B] Melting temperatures are shown for truncated protein M from M. genitalium (MG WT) and M. pneumoniae (MP WT), as well as for protein M analogs that either improve their melting temperature by at least one degree over MG WT (A) or maintain the melting temperature of MG WT (B). Melting temperatures were determined by differential scanning fluorometry (NanoDSF) for WT and mutant protein analogs produced by small-scale protein production from E. coli. The number of mutations and their corresponding amino acid substitutions are listed on the right. The analogs exhibit varying thermal stability, and multiple mutations produce additive effects, increasing the melting temperature. [Figure 16] Example data are shown using differential scanning fluorography (DSF) to measure the melting temperatures of MG WT and MG29. Melting temperatures were determined for all analogs, and results are shown for MG WT (Tm = 41.9 °C) and MG29 (Tm = 55.2 °C). The inflection point of the first derivative indicates the melting temperature (Tm). Proteins were recombinantly expressed in E. coli and purified using nickel column chromatography prior to measurement. [Figures 17A-17C]Figure 1 shows the soluble fraction of Protein M analogs as determined by SDS-PAGE. Seven aliquots (0.4 mg / mL) of each protein were incubated at 37°C for different times. The precipitated protein was pelleted by centrifuging the samples at 15,000 x g for 10 minutes, after which the soluble fraction was run on an SDS-PAGE gel. The proteins were heated for 0, 1, 4, 24, 48, 72, or 96 hours before evaluation. The results show that MG WT (A) begins to precipitate out of solution after 1 to 4 hours of heating. MG27 (B) and MG29 (A) remain soluble for 72 hours, and MG31 (B) and MG40 (C) remain soluble for 24 hours. [Figures 18A-18C]Figure 1 shows that different Protein M analogs block pooled human intravenous immunoglobulin gamma (IVIG) to prevent neutralization of AAV2-luciferase vectors during an in vitro neutralization assay: comparison of 4°C vs. 37°C heat exposure for 1 hour (A and B) or 24 hours (C) of incubation. Protein M vs. IVIG at a 4:1 ratio. Relative light output produced by luciferase activity was measured from cell cultures 24 hours after transduction with an AAV2-luciferase reporter vector (2E8vg) performed in triplicate in a 96-well plate format. Results were normalized to wells containing only AAV2 and phosphate-buffered saline (PBS) (white bars). AAV2 incubated with IVIG (12 μg) for 1 hour showed nearly complete neutralization of AAV. Protein M analog (16 μg) incubated at 4°C prevented AAV neutralization by IVIG when incubated with AAV for 1 hour. This was compared to Protein M analogs incubated at 4°C without IVIG (not performed for MG8 and MG24). When the analogs were heat-exposed at 37°C before incubation with IVIG, MG WT and some analogs with lower melting temperatures did not protect AAV from neutralization, whereas other analogs with higher Tms maintained their ability to protect AAV from neutralization. Most mutant MG analogs maintained their ability to block neutralizing antibodies after 1 hour of exposure at 37°C. However, MG27, MG29, and MG46 prevented AAV neutralization after 24 hours of exposure at 37°C. [Figure 19]We demonstrate that MG WT blocks NAb upon AAV rechallenge one month after primary AAV administration. Wild-type C57BL6 female mice were immunized intraperitoneally with AAV8-GFP (1E9vg), and serum was collected one month later to assess AAV neutralizing antibody titers in vitro. Mice were then intramuscularly administered an AAV8-luciferase reporter vector (1E9vg / leg), where AAV was formulated as a simple mixture with MG WT (33µg / leg) for the right leg of the mice, or formulated with phosphate-buffered saline as a vehicle control for the left leg of the mice. Luminescence imaging of leg muscles was performed two weeks after AAV8-luciferase administration. Three mice with an AAV neutralizing antibody titer of 1:10 showed neutralization of the AAV luciferase reporter vector in the saline-formulated leg, but the AAV luciferase reporter vector was protected from neutralizing antibodies in the MG WT-formulated leg of the same mice. Neutralization of AAV in the saline-formulated leg resulted in an 80% lower mean luciferase signal than in the MG WT-formulated leg. This result demonstrates that AAV can be successfully re-administered with Protein M after the production of neutralizing antibodies induced by a previous AAV administration. [Figure 20]We demonstrate that MG29 blocks NAb upon AAV rechallenge one month after primary AAV administration. Wild-type C57BL6 female mice were immunized intraperitoneally with AAV8-GFP (5E8vg), and serum was collected one month later to assess AAV neutralizing antibody titers in vitro. Mice were then intramuscularly administered an AAV8-luciferase reporter vector (2E9vg / leg), where AAV was formulated as a simple mixture with the modified analog MG29 (500µg / leg) for the right leg, or formulated with phosphate-buffered saline as a vehicle control for the left leg. Luminescence imaging of leg muscles was performed two weeks after AAV8-luciferase administration. Three mice, each with an AAV neutralizing antibody titer of less than 1:100 (1:8, 1:32, and 1:64, respectively), showed neutralization of the AAV luciferase reporter vector in the saline-formulated leg, but the AAV luciferase reporter vector was protected from neutralizing antibodies in the MG29-formulated leg of the same mice. Neutralization of AAV in the saline-formulated leg resulted in a mean luciferase signal that was at least 98% lower than in the MG WT-formulated leg. This result demonstrates that AAV can be successfully re-administered using modified Protein M analogs after the production of neutralizing antibodies induced by a previous AAV administration. [Figure 21] This shows that engineered Protein M analogs exhibit different affinities for IgG. The affinity of specific Protein M analogs was measured using biolayer interferometry (BLI). Binding constants (KD) were calculated using association and dissociation rates (kinetic analysis) over a Protein M concentration range of 15.6 nM to 250 nM. Results show increased or decreased affinity for IgG attached to BLI probes compared to MG WT. [Figure 22] An example of BLI affinity data generated by kinetic analysis is shown. The curve is used to predict kinetic KD values. The curve fit is overlaid on the collected data. [Figure 23]The success rate of mutant stabilization is shown. It represents the frequency at which mutations predicted by Rosetta for MG WT increased (Tm+1°C), decreased (Tm-1°C), or had no effect on stability. "Combinatorial mutations" represent constructs constructed by merging mutant constructs that individually stabilized MG WT. [Figure 24] Conservation of the MP WT sequence is shown. The homology model of MP WT is shown in two orientations and colored by conservation relative to the MG WT sequence according to the BLOSUM62 matrix. Residues are colored according to the degree of conservation, ranging from white (identical) to black (significantly different). Homology levels were constructed using PDB ID: 4NZR as a template. [Figure 25] We demonstrate that codon optimization of the Protein M DNA sequence results in higher production yields. DNA plasmids encoding the MG WT protein were produced using bacterial codons native to MG281 (original PM) or codons optimized for both bacterial and human codon usage (optimized PM). Three pooled bacterial colonies transformed with equivalent concentrations of the original or optimized PM plasmids were cultured and propagated in equivalent volumes of growth medium (10 mL) for equivalent overnight periods. Bacteria were pelleted and crude lysates were produced by freeze-thawing and lysis in equivalent volumes of lysis buffer. The crude lysates were centrifuged, and the supernatants were collected for protein separation on SDS-PAGE gels. Equal volumes from each lysate were run on an SDS-PAGE gel and transferred to a Western blot, which was then probed with a mouse antibody against the 6x histidine tag present at the amino terminus of MG WT, followed by a goat anti-mouse secondary antibody conjugated to horseradish peroxidase (HRP). The band intensity of the MG WT protein was measured using a luminol chemiluminescence assay. The resulting MG WT yield was calculated based on the band intensity of Western blots normalized to the bacterial pellet weight. The optimized PM plasmid resulted in a nearly four-fold increase in MG WT protein yield compared to the original PM plasmid. [Figure 26] The improved pH stability of the mutants is shown. Melting temperatures (Tm) were determined using NanoDSF at different pH conditions. The protein was purified via SEC in PBS and buffer-exchanged into glycine (pH 2.5 & 3.5), acetate (pH 4.5 & 5.5), and phosphate (pH 6.5 and 7.5) buffers. [Figures 27A-27D] Figure 1 shows a sequence alignment of wild-type M. genitalium protein M amino acids 74 to 479 (SEQ ID NO: 3) and engineered protein M MG1 (SEQ ID NO: 4), MG8 (SEQ ID NO: 5), MG13 (SEQ ID NO: 6), MG15 (SEQ ID NO: 7), MG21 (SEQ ID NO: 8), MG22 (SEQ ID NO: 9), MG23 (SEQ ID NO: 10), MG24 (SEQ ID NO: 11), MG27 (SEQ ID NO: 12), MG28 (SEQ ID NO: 13), MG29 (SEQ ID NO: 14), MG31 (SEQ ID NO: 15), MG33 (SEQ ID NO: 16), MG38 (SEQ ID NO: 17), MG40 (SEQ ID NO: 18), MG43 (SEQ ID NO: 19), MG44 (SEQ ID NO: 20), MG45 (SEQ ID NO: 21), and MG46 (SEQ ID NO: 22). [Figure 28] 1 shows a sequence alignment of wild-type M. genitalium protein M amino acids 74-479 (SEQ ID NO: 3) and the corresponding fragment of M. pneumoniae protein M (SEQ ID NO: 23). DETAILED DESCRIPTION OF THE INVENTION

[0023] The present invention is described in more detail below. This description is not intended to be a detailed catalog of all the different ways in which the invention may be practiced or all the features that may be added to the invention. For example, features described with respect to one embodiment may be incorporated into other embodiments, and features described with respect to a particular embodiment may be omitted from that embodiment. Moreover, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of this disclosure without departing from the invention. Therefore, the following specification is intended to describe some particular embodiments of the invention, but is not intended to exhaustively specify all permutations, combinations, and variations thereof.

[0024] It is expressly intended that the various features of the invention described herein may be used in any combination unless the context dictates otherwise. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein may be excluded or omitted. By way of example, if the specification states that a composite comprises components A, B, and C, it is expressly intended that any of A, B, or C, or combinations thereof, singly or in any combination, may be omitted and negated.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the present invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present invention.

[0026] Nucleotide sequences are presented herein in a single strand only, in a 5' to 3' orientation, left to right, unless expressly indicated otherwise. Nucleotides and amino acids are represented herein in the format recommended by the IUPAC-IUB Biochemical Nomenclature Commission, or (for amino acids) by either the single-letter code or the three-letter code, both in accordance with 37 CFR § 1.822 and established usage.

[0027] Unless otherwise indicated, standard methods known to those of skill in the art can be used for the production of recombinant and synthetic polypeptides, antibodies or antigen-binding fragments thereof, the manipulation of nucleic acid sequences, the production of transformed cells, the construction of rAAV constructs, modified capsid proteins, packaging vectors expressing AAV rep and / or cap sequences, and transiently and stably transfected packaging cells. Such techniques are known to those of skill in the art. See, e.g., SAMBROOK et al., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, NY, 1989); FMAUSUBEL et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0028] All publications, patent applications, patents, nucleotide sequences, amino acid sequences, and other references mentioned herein are incorporated by reference in their entirety.

[0029] definition As used in the description of this invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0030] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, and, when interpreted in the alternative ("or"), the absence of combinations.

[0031] Furthermore, the present invention contemplates that in some embodiments of the invention, any feature or combination of features set forth herein may be excluded or omitted.

[0032] Additionally, as used herein, the term "about," when referring to a measurable value such as an amount, dose, time, temperature, etc. of a compound or agent of the invention, is meant to encompass variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the stated amount.

[0033] As used herein, the transitional phrase "consisting essentially of" should be interpreted to include the recited materials or steps and those that do not materially affect the basic and novel feature(s) of the claimed invention. Thus, as used herein, the term "consisting essentially of" should not be interpreted as equivalent to "comprising."

[0034] The term "consists essentially of" (and grammatical variations) when applied to a polynucleotide or polypeptide sequence of the invention refers to a polynucleotide or polypeptide consisting of both a recited sequence (e.g., SEQ ID NO:) and a total of 10 or fewer (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) additional nucleotides or amino acids on the 5' and / or 3' or N-terminus and / or C-terminus of the recited sequence or between the two termini (e.g., between domains), such that the function of the polynucleotide or polypeptide is not substantially altered. A total of 10 or fewer additional nucleotides or amino acids includes the total number of additional nucleotides or amino acids taken together. The term "substantially altered" when applied to a polynucleotide of the invention refers to an increase or decrease in the ability to express the encoded polypeptide by at least about 50% or more compared to the expression level of a polynucleotide consisting of the recited sequence. The term "substantially altered" when applied to a polypeptide of the invention refers to an increase or decrease in biological activity by at least about 50% or more compared to the activity of a polypeptide consisting of the recited sequence.

[0035] The term "parvovirus" as used herein encompasses the Parvoviridae family and includes autonomously replicating parvoviruses and dependoviruses. Autonomous parvoviruses include members of the Parvovirus, Erythrovirus, Densovirus, Iteravirus, and Contravirus genera. Exemplary autonomous parvoviruses include, but are not limited to, minute virus of mice, bovine parvovirus, canine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, H1 parvovirus, Muscovy duck parvovirus, snake parvovirus, and B19 virus. Other autonomous parvoviruses are known to those skilled in the art. See, e.g., FIELDS et al., VIROLOGY, Vol. 2, Chapter 69 (4th ed., Lippincott-Raven Publishers).

[0036] The Dependovirus genus includes adeno-associated viruses (AAVs), including, but not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, avian AAV, bovine AAV, canine AAV, caprine AAV, snake AAV, equine AAV, and ovine AAV. See, e.g., FIELDS et al., VIROLOGY, Vol. 2, Chapter 69 (4th ed., Lippincott-Raven Publishers); and Table 1.

[0037] The term "adeno-associated virus" (AAV) in the context of the present invention includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, and ovine AAV, as well as any other AAV now known or later discovered. See, e.g., Bernard N. Fields et al., VIROLOGY, Vol. 2, Chapter 69 (4th ed., Lippincott-Raven Publishers). Several additional AAV serotypes and clades have been identified (see, e.g., Gao et al., (2004) J. Virol. 78:6381-6388 and Table 1), and are also encompassed by the term "AAV."

[0038] The parvovirus particles and genomes of the present invention may be derived from, but are not limited to, AAV. The genome sequences of various serotypes of AAV and autonomous parvoviruses, as well as the sequences of native ITRs, Rep proteins, and capsid subunits, are known in the art. Such sequences can be found in the literature or public databases such as GenBank. For example, GenBank accession numbers NC_002077, NC_001401, NC_001729, NC_001863, NC_001829, NC_001862, NC_000883, NC_001701, NC_001510, NC_006152, NC_006261, AF063497, U89790, AF043303, AF028705, AF028704, J02275, J019 01, J02275, X01457, AF288061, AH009962, AY028226, AY028223, AY631966, AX753250, EU285562, NC_001358, NC_001540, AF513851, AF513852 and AY530579; the disclosures of which are incorporated by reference herein for their teaching of parvovirus and AAV nucleic acid and amino acid sequences.Also, for example, Bantel-Schaal et al.,(1999)J.Virol.73:939;Chiorini et al.,(1997)J.Virol.71:6823;Chiorini et al.,(1999)J.Virol.73:1309;Gao et al.,(2002)Proc.Nat.Acad.Sci.USA 99:11854;Moris et al.,(2004)Virol.33-:375-383;Mori et al.,(2004)Virol.330:375;Muramatsu et al.,(1996)Virol.221:208;Ruffing et al. al.,(1994)J.Gen.Virol.75:3385;Rutledge et See also, e.g., (1998) J. Virol. 72:309; Schmidt et al., (2008) J. Virol. 82:8911; Shade et al., (1986) J. Virol. 58:921; Srivastava et al., (1983) J. Virol. 45:555; Xiao et al., (1999) J. Virol. 73:3994; International Patent Publications WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; and U.S. Patent No. 6,156,303, the disclosures of which are incorporated herein by reference for their teaching of parvovirus and AAV nucleic acid and amino acid sequences. See also, Table 1. An early description of AAV1, AAV2, and AAV3 ITR sequences is provided by Xiao, X., (1996), "Characterization of Adeno-associated virus (AAV) DNA replication and integration," Ph.D. dissertation, University of Pittsburgh, Pittsburgh, PA (incorporated herein in its entirety).

[0039] A "chimeric" AAV nucleic acid capsid coding sequence or AAV capsid protein combines portions of two or more capsid sequences. A "chimeric" AAV virion or particle comprises a chimeric AAV capsid protein.

[0040] As used herein, the term "tropism" refers to the preferential, but not necessarily exclusive, entry of a vector (e.g., a viral vector) into a particular cell or tissue type(s) and / or a preferential, but not necessarily exclusive, interaction with a cell surface that facilitates entry into a particular cell or tissue type, optionally and preferably followed by expression (e.g., transcription, translation) of sequences carried by the vector contents (e.g., the viral genome) in the cell, e.g., for recombinant viruses, expression of a heterologous nucleotide sequence(s). Those skilled in the art understand that transcription of heterologous nucleic acid sequences from the viral genome cannot be initiated in the absence of trans-acting factors, e.g., for an inducible promoter or otherwise regulated nucleic acid sequence. In the case of the rAAV genome, gene expression from the viral genome can be from a stably integrated provirus and / or from a non-integrated episome, as well as any other form that viral nucleic acid may take within a cell.

[0041] The term "tropism profile" refers to the transduction pattern of one or more target cells, tissues, and / or organs. A representative example of a chimeric AAV capsid has a tropism profile characterized by efficient transduction of cells in the central nervous system (CNS) with only minor transduction of peripheral organs (see, for example, U.S. Patent No. 9,636,370 (McCown et al.) and U.S. Patent Publication No. 2017 / 0360960 (Gray et al.)). A vector (e.g., a viral vector, e.g., an AAV capsid) that expresses a specific tropism profile can be referred to as "tropic" for its tropism profile, such as neurotropism, hepatotropism, etc.

[0042] As used herein, "transduction" of a cell by a viral vector (e.g., an AAV vector) refers to the entry of the vector into the cell and the transfer of genetic material into the cell by incorporation of nucleic acid into the viral vector and subsequent transfer into the cell via the viral vector.

[0043] JPEG2026000908000002.jpg223166

[0044] Unless otherwise indicated, "efficient transduction" or "efficient tropism" or similar terms can be determined by reference to appropriate positive or negative controls (e.g., at least about 50%, 60%, 70%, 80%, 85%, 90%, 95% or more of the transduction or tropism, respectively, of a positive control, or at least about 110%, 120%, 150%, 200%, 300%, 500%, 1000% or more of the transduction or tropism, respectively, of a negative control).

[0045] Similarly, by reference to appropriate controls, one can determine whether a virus "does not transduce efficiently" or "does not have efficient tropism" (or similar terms) for a target tissue. In certain embodiments, a viral vector does not efficiently transduce (i.e., does not have efficient tropism for) tissues other than the CNS, such as the liver, kidney, gonads, and / or germ cells. In certain embodiments, undesired transduction of tissue(s) (e.g., liver) is 20% or less, 10% or less, 5% or less, 1% or less, 0.1% or less of the transduction level of the desired target tissue(s) (e.g., CNS cells).

[0046] The terms "5' portion" and "3' portion" are relative terms used to define the spatial relationship between two or more elements. Thus, for example, the "3' portion" of a polynucleotide refers to a segment of the polynucleotide that is downstream of another segment. The term "3' portion" is not intended to indicate that a segment is necessarily at the 3' end of the polynucleotide, or even necessarily in the 3' half of the polynucleotide, although it may be. Similarly, the "5' portion" of a polynucleotide refers to a segment of the polynucleotide that is upstream of another segment. The term "5' portion" is not intended to indicate that a segment is necessarily at the 5' end of the polynucleotide, or even necessarily in the 5' half of the polynucleotide, although it may be.

[0047] As used herein, the term "polypeptide" includes both peptides and proteins, unless otherwise indicated.

[0048] A "polynucleotide," "nucleic acid," or "nucleotide sequence" may be RNA, DNA, or a DNA-RNA hybrid sequence (containing both naturally occurring and non-naturally occurring nucleotides), but is preferably either a single-stranded or double-stranded DNA sequence.

[0049] The term "regulatory element" refers to a genetic element that controls some aspects of the expression of a nucleic acid sequence. For example, a promoter is a regulatory element that facilitates the initiation of transcription of an operably linked codon region. Other regulatory elements are splicing signals, polyadenylation signals, termination signals, etc. A region within a nucleic acid sequence or polynucleotide in which one or more regulatory elements are found can be referred to as a "regulatory region."

[0050] The term "fragment" as applied to a polypeptide is understood to mean an amino acid sequence of reduced length compared to a reference polypeptide or amino acid sequence that comprises, consists essentially of, and / or consists of an amino acid sequence of consecutive amino acids identical to the reference polypeptide or amino acid sequence. In some embodiments, such fragments can comprise, consist essentially of, and / or consist of a peptide having a length of at least about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, or more consecutive amino acids of a polypeptide or amino acid sequence of the invention. In some embodiments, such fragments can comprise, consist essentially of, and / or consist of peptides having a length of less than about 4, 6, 8, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or 500 consecutive amino acids of a polypeptide or amino acid sequence of the present invention.

[0051] As used herein, a "functional fragment" refers to one that substantially maintains at least one biological activity normally associated with the polypeptide (e.g., antibody binding). A "functional fragment" substantially maintains all of the activities of the unmodified polypeptide. "Substantially maintaining" biological activity means that the polypeptide maintains at least about 20%, 30%, 40%, 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more of the biological activity of the native polypeptide (and may have a higher activity level than the native polypeptide). A "non-functional" polypeptide refers to one that exhibits little or essentially no detectable biological activity normally associated with the polypeptide (e.g., at most, only a slight amount, e.g., less than about 10% or even 5%). Biological activity, such as antibody binding, can be measured using assays well known in the art and described herein.

[0052] As used herein, the term "operably linked" with respect to nucleic acids refers to a functional linkage between two or more nucleic acids. For example, a promoter sequence may be described as "operably linked" to a heterologous nucleic acid sequence because the promoter sequence initiates and / or mediates transcription of the heterologous nucleic acid sequence. In some embodiments, operably linked nucleic acid sequences are contiguous and / or in the same reading frame.

[0053] As used herein, the term "open reading frame (ORF)" refers to a portion of a polynucleotide (e.g., a gene) that encodes a polypeptide and includes an initiation start site (i.e., a Kozak sequence) from which transcription of the polypeptide begins. The term "coding region" can be used interchangeably with open reading frame.

[0054] As used herein, the term "codon optimization" refers to a gene coding sequence that has been optimized to increase expression by replacing one or more codons normally present in the coding sequence (e.g., including the wild-type sequence, e.g., the coding sequence for protein M) with codons for the same (synonymous) amino acid. In this manner, the proteins encoded by the genes are identical, but the underlying nucleobase sequences of the genes or the corresponding mRNAs are different. In some embodiments, optimization replaces one or more rare codons (i.e., codons for tRNAs that occur relatively rarely in cells from a particular species) with more frequently occurring synonymous codons to improve the efficiency of translation. For example, in human codon optimization, one or more codons in the coding sequence are replaced with codons that occur more frequently in human cells for the same amino acid. Codon optimization can also increase gene expression through other mechanisms that may improve the efficiency of transcription and / or translation. Strategies include, but are not limited to, increasing the total GC content (i.e., the percent of guanine and cytosine in the entire coding sequence), reducing the CpG content (i.e., the number of CG or GC dinucleotides in the coding sequence), removing potential splice donor or acceptor sites, and / or adding or removing ribosome entry and / or start sites, such as Kozak sequences. Desirably, the codon-optimized gene exhibits improved protein expression, e.g., the protein encoded thereby is expressed at a detectably greater level in a cell compared to the protein expression level provided by the wild-type gene in an otherwise similar cell. Codon optimization also provides the ability to distinguish the codon-optimized gene and / or corresponding mRNA from the endogenous gene and / or corresponding mRNA in vitro or in vivo.

[0055] As used herein, the term "sequence identity" has its standard meaning in the art. As is known in the art, many different programs can be used to identify whether a polynucleotide or polypeptide has sequence identity or similarity to a known sequence. Sequence identity or similarity can be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the sequence identity alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), by the BestFit sequence program described by Devereux et al., Nucl. Acid Res. 12:387 (1984), preferably using the default settings, or by inspection.

[0056] An example of a useful algorithm is PILEUP, which creates a multiple sequence alignment from a group of related sequences using progressive pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351 (1987); the method is similar to that described by Higgins & Sharp, CABIOS 5:151 (1989).

[0057] Another example of a useful algorithm is the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215:403 (1990) and Karlin et al., Proc. Natl. Acad. Sci. USA 90:5873 (1993). A particularly useful BLAST program is the WU-BLAST-2 program, Altschul et al., Meth. Enzymol., 266:460 (1996); obtained from blast.wustl / edu / blast / README.html. WU-BLAST-2 uses several search parameters, which are preferably set to default values. The parameters are dynamic values ​​and are established by the program itself depending on the composition of the particular sequence and the composition of the particular database in which the sequence of interest is being searched; however, values ​​may be adjusted to increase sensitivity.

[0058] Another useful algorithm is gapped BLAST, as reported by Altschul et al., Nucleic Acids Res. 25:3389 (1997).

[0059] Percentage amino acid sequence identity values ​​are determined by dividing the number of matching identical residues by the total number of residues in the "longer" sequence within the aligned region, the "longer" sequence being the one with the most actual residues within the aligned region (ignoring gaps introduced by WU-Blast-2 to maximize the alignment score).

[0060] In a similar manner, percent nucleic acid sequence identity is defined as the percentage of nucleotide residues in a candidate sequence that are identical with nucleotides in a polynucleotide specifically disclosed herein.

[0061] Alignment may include the introduction of gaps in the aligned sequences.Furthermore, it is understood that for sequences that contain more or fewer nucleotides than the polynucleotides specifically disclosed herein, in one embodiment, the percentage of sequence identity is determined based on the number of identical nucleotides relative to the total number of nucleotides.Thus, for example, the sequence identity of a sequence that is shorter than the sequences specifically disclosed herein, in one embodiment, is determined using the number of nucleotides in the shorter sequence.In calculating percent identity, relative weight is not assigned to various occurrences of sequence variation, such as insertion, deletion, substitution, etc.

[0062] In one embodiment, only identities are scored positively (+1) and all forms of sequence variation, including gaps, are assigned a value of "0," obviating the need for the weighting scale or parameters described below for sequence similarity calculations. Percent sequence identity can be calculated, for example, by dividing the number of matching identical residues by the total number of residues of the "shorter" sequence in the aligned region and multiplying by 100. The "longer" sequence is the one with the most actual residues in the aligned region.

[0063] As used herein, an "isolated" nucleic acid or nucleotide sequence (e.g., "isolated DNA" or "isolated RNA") means a nucleic acid or nucleotide sequence that is separated from or substantially free of at least some of the other components of the organism or virus in which it naturally occurs, e.g., a structural component of a cell or virus or other polypeptides or nucleic acids that are normally found in association with the nucleic acid or nucleotide sequence.

[0064] Similarly, an "isolated" polypeptide means a polypeptide that is separated from or substantially free of at least some of the other components of the organism or virus in which it naturally originates, e.g., other polypeptides or nucleic acids that are normally found in association with the polypeptide, e.g., structural components or components of a cell or virus.

[0065] The term "modified," as used herein, as applied to a polynucleotide or polypeptide sequence, refers to a sequence that differs from the wild-type sequence due to one or more deletions, additions, substitutions, or any combination thereof.

[0066] As used herein, by "isolating" a viral vector (or grammatical equivalents) it is meant that the viral vector is at least partially separated from at least some of the other components in the starting material.

[0067] By the terms "treat," "treating," or "treatment of" (or grammatical equivalents) is meant to reduce the severity of, or at least partially improve or ameliorate, the condition of a subject, and / or to alleviate, relieve, or reduce at least one clinical symptom, and / or to slow the progression of the condition.

[0068] As used herein, the terms "prevent," "prevents," or "prevention" (and grammatical equivalents) mean to delay or inhibit the onset of a disease. The term does not require complete elimination of the disease, but encompasses any type of prophylactic treatment to reduce the incidence of symptoms or delay the onset of symptoms.

[0069] As used herein, a "treatment effective" amount is an amount sufficient to provide some improvement or benefit to the subject. In other words, a "treatment effective" amount is an amount that provides some relief, alleviation, reduction, or stabilization of at least one clinical symptom in the subject. Those skilled in the art understand that the therapeutic effect need not be complete or curative, as long as some benefit is provided to the subject.

[0070] As used herein, a "prophylactically effective" amount is an amount sufficient to prevent and / or delay the onset of a disease, disorder, and / or clinical symptom in a subject, and / or reduce and / or delay the severity of the onset of a disease, disorder, and / or clinical symptom in a subject compared to that which would occur in the absence of the method of the present invention. One of skill in the art will understand that the level of prevention need not be complete, as long as some benefit is provided to the subject.

[0071] A "heterologous nucleotide sequence" or "heterologous nucleic acid," with respect to a virus, is a sequence or nucleic acid, respectively, that does not naturally occur in the virus. Generally, a heterologous nucleic acid or nucleotide sequence comprises an open reading frame encoding a polypeptide and / or untranslated RNA.

[0072] A "vector" refers to a compound used as a vehicle to transport foreign genetic material into another cell where it can be replicated and / or expressed. A cloning vector containing a foreign nucleic acid is called a recombinant vector. Examples of nucleic acid vectors are plasmids, viral vectors, cosmids, expression cassettes, and artificial chromosomes. Recombinant vectors typically contain an origin of replication, a multiple cloning site, and a selectable marker. A nucleic acid sequence typically consists of an insert (recombinant nucleic acid or transgene) and a larger sequence that serves as the "backbone" of the vector. The purpose of a vector to transfer genetic information to another cell is typically to isolate, propagate, or express the insert in the target cell. An expression vector (expression construct or expression cassette) is intended for the expression of exogenous genes in target cells and generally contains a promoter sequence that drives the expression of the exogenous gene / ORF. Insertion of a vector into a target cell is called transformation or transfection for bacterial and eukaryotic cells, while insertion of a viral vector is often called transduction. The term "vector" may also be used generally to describe an item that serves to carry foreign genetic material into another cell, such as, but not limited to, a cell to be transformed or a nanoparticle.

[0073] As used herein, the terms "vector," "viral vector," "delivery vector" (and similar terms) generally refer to viral particles that, in certain embodiments, function as nucleic acid delivery vehicles and contain viral nucleic acid (i.e., vector genomes) packaged within the virion. Viral vectors of the present invention comprise chimeric AAV capsids of the present invention and can package AAV or rAAV genomes or any other nucleic acid (including viral nucleic acids). Alternatively, in some contexts, the terms "vector," "viral vector," "delivery vector" (and similar terms) may be used to refer to virions that act as vehicles for delivering vector genomes (e.g., vDNA) absent from the virion and / or viral capsids that act as vehicles for delivering molecules tethered to or packaged within the capsid.

[0074] The viral vectors of the invention may also be double parvovirus particles as described in International Patent Publication WO 01 / 92551, the disclosure of which is incorporated herein by reference in its entirety. Thus, in some embodiments, a double-stranded (duplex) genome may be packaged.

[0075] A "recombinant AAV vector genome" or "rAAV genome" is an AAV genome (i.e., vDNA) containing at least one inverted terminal repeat (e.g., one, two, or three inverted terminal repeats) and one or more heterologous nucleotide sequences. rAAV vectors generally retain the 145-base terminal repeat(s) (TR(s)) in cis to produce virus, although modified AAV TRs and non-AAV TRs containing partially or completely synthetic sequences can also serve this purpose. All other viral sequences are not required and may be supplied in trans (Muzyczka, (1992) Curr. Topics Microbiol. Immunol. 158:97). rAAV vectors may also contain two TRs (e.g., AAV TRs), which are generally at the 5' and 3' ends of the heterologous nucleotide sequence(s), but need not be contiguous with them. The TRs may be identical to or different from each other. The vector genome may contain a single ITR at its 3' or 5' end.

[0076] The term "terminal repeat" or "TR" includes any viral or synthetic terminal repeat sequence that forms a hairpin structure and functions as an inverted terminal repeat (ITR) (i.e., mediates a desired function, such as replication, viral packaging, integration, and / or proviral rescue). The TR may be an AAV ITR or a non-AAV TR. For example, non-AAV TR sequences, such as those of other parvoviruses (e.g., canine parvovirus (CPV), mouse parvovirus (MVM), and human parvovirus B-19), or the SV40 hairpin that acts as the SV40 origin of replication, can be used as a TR, which may be further modified by truncation, substitution, deletion, insertion, and / or addition. Furthermore, the TR may be partially or completely synthetic, e.g., the "double-D sequence" described in U.S. Patent No. 5,478,745 (Samulski et al.).

[0077] Parvovirus genomes have palindromic sequences at both their 5' and 3' ends. The palindromic nature of the sequences results in the formation of a hairpin structure stabilized by the formation of hydrogen bonds between complementary base pairs. This hairpin structure is thought to take a "Y" or "T" shape. See, e.g., FIELDS et al., VIROLOGY, Vol. 2, Chapters 69 & 70 (4th ed., Lippincott-Raven Publishers).

[0078] "AAV inverted terminal repeats" or "AAV ITRs" may be derived from any AAV, including, but not limited to, serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11, or any other AAV now known or later discovered (see, e.g., Table 1). AAV ITRs need not have native ITR sequences (e.g., native AAV ITR sequences may be altered by insertions, deletions, truncations, and / or missense mutations), so long as the ITRs mediate the desired function, such as replication, viral packaging, integration, and / or proviral rescue.

[0079] The terms "rAAV particle" and "rAAV virion" are used interchangeably herein. An "rAAV particle" or "rAAV virion" comprises an rAAV vector genome packaged within an AAV capsid.

[0080] The viral vectors of the present invention may further be "targeted" viral vectors (e.g., with directed tropism) and / or "hybrid" parvoviruses (i.e., in which the viral ITRs and the viral capsid are derived from different parvoviruses) as described in International Patent Publication WO 00 / 28004 and Chao et al., (2000) Mol. Therapy 2:619.

[0081] Additionally, the viral capsid or genomic elements may contain other modifications, including insertions, deletions and / or substitutions.

[0082] As used herein, the term "amino acid" encompasses any naturally occurring amino acid, modified forms thereof, and synthetic amino acids, including non-naturally occurring amino acids.

[0083] Naturally occurring, levorotatory (L-) amino acids are listed in Table 2. JPEG2026000908000003.jpg194166

[0084] Alternatively, the amino acid may be a modified amino acid residue (non-limiting examples are shown in Table 3) or may be an amino acid modified by post-translational modification (e.g., acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation, or sulfation).

[0085] JPEG2026000908000004.jpg238140

[0086] Additionally, non-naturally occurring amino acids can be "unnatural" amino acids as described by Wang et al. (2006) Annu. Rev. Biophys. Biomol. Struct. 35:225-49. These non-natural amino acids can be advantageously used to chemically link a molecule of interest to an AAV capsid protein.

[0087] Conservative amino acid substitutions are known in the art. In certain embodiments, conservative amino acid substitutions include substitutions within one or more of the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and / or phenylalanine, tyrosine.

[0088] The terms "template" or "substrate" are used herein to refer to a polynucleotide sequence that can be replicated to produce parvovirus viral DNA. For vector production purposes, the template is typically embedded within a larger nucleotide sequence or construct, including, but not limited to, a plasmid, naked DNA vector, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), or viral vector (e.g., adenovirus, herpesvirus, Epstein-Barr virus, AAV, baculovirus, retroviral vector, etc.). Alternatively, the template can be stably integrated into the chromosome of the packaging cell.

[0089] As used herein, a parvovirus or AAV "Rep coding sequence" refers to a nucleic acid sequence that encodes the parvovirus or AAV nonstructural proteins that mediate viral replication and the production of new virus particles. Parvovirus and AAV replication genes and proteins are described, for example, in FIELDS et al., VIROLOGY, Vol. 2, Chapters 69 & 70 (4th ed., Lippincott-Raven Publishers).

[0090] A "Rep coding sequence" need not encode all of the parvovirus or AAV Rep proteins. For example, with respect to AAV, the Rep coding sequence need not encode all four AAV Rep proteins (Rep78, Rep68, Rep52, and Rep40); indeed, AAV5 is believed to express only the spliced ​​Rep68 and Rep40 proteins. In representative embodiments, the Rep coding sequence encodes at least the replication proteins necessary for viral genome replication and packaging into new virions. The Rep coding sequence generally encodes at least one large Rep protein (i.e., Rep78 / 68) and one small Rep protein (i.e., Rep52 / 40). In certain embodiments, the Rep coding sequence encodes the AAV Rep78 protein and the AAV Rep52 and / or Rep40 protein. In other embodiments, the Rep coding sequence encodes the Rep68 and Rep52 and / or Rep40 proteins. Moreover, in further embodiments, the Rep coding sequence encodes Rep68 and Rep52 proteins, Rep68 and Rep40 proteins, Rep78 and Rep52 proteins, or Rep78 and Rep40 proteins.

[0091] As used herein, the term "large Rep protein" refers to Rep68 and / or Rep78. The large Rep proteins of the claimed invention can be either wild-type or synthetic. Wild-type large Rep proteins can be from any parvovirus or AAV, including, but not limited to, serotypes 1, 2, 3a, 3b, 4, 5, 6, 7, 8, 9, 10, 11, or 13, or any other AAV now known or later discovered (see, e.g., Table 1). Synthetic large Rep proteins can be altered by insertions, deletions, truncations, and / or missense mutations.

[0092] Those skilled in the art will further understand that replication proteins do not need to be encoded by the same polynucleotide. For example, with respect to MVM, the NS-1 and NS-2 proteins (splice variants) can be expressed independently of each other. Similarly, with respect to AAV, the p19 promoter can be inactivated, the large Rep protein(s) can be expressed from one polynucleotide, and the small Rep protein(s) can be expressed from a different polynucleotide. However, it is typically more convenient to express replication proteins from a single construct. In some systems, the viral promoter (e.g., the AAV p19 promoter) may not be recognized by the cell, and therefore it is necessary to express the large Rep protein and the small Rep protein from separate expression cassettes. In other instances, it may be desirable to express the large Rep protein and the small Rep protein separately, i.e., under the control of separate transcriptional and / or translational control elements. For example, it may be desirable to control the expression of the large Rep protein so as to reduce the ratio of the large to the small Rep protein. In the case of insect cells, it may be advantageous to downregulate the expression of large Rep proteins (e.g., Rep78 / 68) to avoid toxicity to the cells (see, e.g., Urabe et al., (2002) Human Gene Therapy 13:1935).

[0093] As used herein, a parvovirus or AAV "cap coding sequence" encodes the structural proteins that form a functional parvovirus or AAV capsid (i.e., capable of packaging DNA and infecting a target cell). Typically, the cap coding sequence encodes all of the parvovirus or AAV capsid subunits, although fewer than all of the capsid subunits may be encoded so long as a functional capsid is produced. Typically, but not necessarily, the cap coding sequence is present on a single nucleic acid molecule.

[0094] The capsid structure of autonomous parvoviruses and AAV is described in detail by Bernard N. Fields et al., VIROLOGY, Vol. 2, Chapters 69 & 70 (4th ed., Lippincott-Raven Publishers).

[0095] By "substantially maintain" a property, it is meant that at least about 75%, 85%, 90%, 95%, 97%, 98%, 99% or 100% of the property (e.g., activity or other measurable characteristic) is maintained.

[0096] Methods of using Protein M and its derivatives for binding to antibodies One aspect of the present invention relates to a method for inhibiting neutralization of a heterologous agent by neutralizing antibodies when the heterologous agent is administered to a subject, comprising the step of administering to the subject an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, thereby inhibiting neutralization of the heterologous agent.

[0097] Another aspect of the present invention relates to a method for expressing a polypeptide or functional nucleic acid in a subject, comprising administering to the subject (a) a nucleic acid delivery vector encoding the polypeptide or functional nucleic acid, and (b) an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, thereby expressing the polypeptide or functional nucleic acid in the subject.

[0098] A further aspect of the present invention relates to a method for editing a gene in a subject, comprising administering to the subject (a) a gene editing complex and (b) an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, thereby expressing a polypeptide or functional nucleic acid in the subject.

[0099] As used herein, the term "heterologous agent" refers to an agent that is not naturally found in the subject to which it is administered. The term also includes recombinant or synthetic versions of agents that are naturally found in the subject. A heterologous agent may be one against which neutralizing antibodies are present in the subject prior to administration of the heterologous agent, or one that may produce neutralizing antibodies upon administration to a subject. A heterologous agent may be one that has never been administered to a subject. A heterologous agent may be one that has been previously administered to a subject.

[0100] As used herein, the term "neutralizing antibody" refers to an antibody that specifically binds to a heterologous agent and inhibits one or more biological activities of the heterologous agent after administration to a subject.

[0101] In some embodiments, the heterologous agent may be a nucleic acid delivery vector, such as a viral vector or a non-viral vector. In some embodiments, the viral vector is an adeno-associated virus, retrovirus, lentivirus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpesvirus, Epstein-Barr virus, or adenovirus vector. In some embodiments, the non-viral vector is a plasmid, liposome, charged lipid, nucleic acid-protein complex, or biopolymer.

[0102] In some embodiments, the heterologous agent is a gene editing complex, such as a CRISPR complex.

[0103] In some embodiments, the heterologous agent is a protein or a nucleic acid. In some embodiments, the protein is an enzyme, a regulatory protein, or a structural protein, for example, one that can be used to replace a missing or defective protein in a subject. In some embodiments, the nucleic acid is a functional nucleic acid, for example, an antisense nucleic acid or an inhibitory RNA.

[0104] An effective amount of Protein M or a functional fragment or derivative thereof is an amount that at least partially blocks inhibition of a heterologous agent by a neutralizing antibody. In some embodiments, an effective amount of Protein M is an amount sufficient to inhibit neutralization by at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%. In some embodiments, an effective amount of Protein M, or a functional fragment or derivative thereof, is sufficient to produce a ratio of Protein M to total immunoglobulin in the subject on a molar basis of about 0.5:1 to about 8:1, or any range therein, e.g., about 0.5:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1, 5.5:1, 6:1, 6.5:1, 7:1, 7.5:1, or 8:1, or any range therein. In some embodiments, the ratio is about 0.5:1 to about 6:1, about 0.5:1 to about 4:1, about 0.5:1 to about 2.5:1, about 0.5:1 to about 2:1, about 1:1 to about 8:1, about 1.5:1 to about 8:1, or about 2:1 to about 8:1. In one embodiment, the ratio is about 1:1 to about 3:1, e.g., about 2:1. Total immunoglobulin can be total serum immunoglobulin (e.g., for systemic administration of Protein M). Total immunoglobulin can be the total level in a localized fluid or tissue (e.g., for specific delivery to the eye, ear, lung, brain, muscle, joint, etc.). Total immunoglobulin can be measured by techniques known in the art, such as by performing ELISA on serum using an antibody that binds to the Fc region of immunoglobulins, or by using Protein A or G, which bind to immunoglobulins. Furthermore, for mice, their serum is known to contain 5 mg / ml to 10 mg / ml of immunoglobulin. The normal range for serum immunoglobulin in humans is 8 to 10 mg / ml. For in vivo estimation, the ratio can be calculated using a high end of 10 mg / ml.Local immunoglobulin content can be estimated based on tissue weight (40 mL serum / kg body weight) or the concentration of Ig in a particular body fluid and, if less than blood serum, the volume of fluid within that organ (e.g., eye, cerebrospinal fluid).

[0105] Protein M can be administered to a subject by any schedule found to be effective in blocking inhibition of the heterologous agent by neutralizing antibodies. In some embodiments, Protein M, or a functional fragment or derivative thereof, is administered to a subject prior to administration of the heterologous agent, e.g., at least about 1, 5, 10, 15, 20, 30, 40, or 50 minutes or at least about 1, 2, 3, 4, 5, 6, 12, 18, or 24 hours prior to administration of the heterologous agent. In some embodiments, Protein M, or a functional fragment or derivative thereof, is administered to a subject simultaneously with administration of the heterologous agent. As used herein, the term "concurrently" means sufficiently close in time to produce a combined effect (i.e., "concurrently" can be "simultaneously" or can refer to two or more events occurring one after the other within a short period of time).

[0106] In some embodiments, the heterologous agent is combined with Protein M or a functional fragment or derivative thereof prior to administration to the subject, e.g., the two components are mixed together in a single composition prior to administration. The heterologous agent may be combined with Protein M or a functional fragment or derivative thereof at least about 1, 5, 10, 15, 20, 30, 40, or 50 minutes or at least about 1, 2, 3, 4, 5, 6, 12, 18, or 24 hours prior to administration to the subject. In other embodiments, Protein M or a functional fragment or derivative thereof and the heterologous agent are administered in separate compositions.

[0107] In some embodiments, it may be necessary to administer the heterologous agent and / or protein M, or a functional fragment or derivative thereof, to a subject more than once to provide a therapeutic or other beneficial effect. Protein M, or a functional fragment or derivative thereof, may be administered, for example, once, twice, three times, four times, or more times. In some embodiments, protein M, or a functional fragment or derivative thereof, is administered to a subject each time a heterologous agent is administered to the subject, e.g., in a manner similar to that described above, e.g., before or currently with the heterologous agent. The use of protein M with each administration of a heterologous agent may inhabit the effect of NAbs against the heterologous agent, which is often problematic upon re-administration. In some embodiments, the same protein M, or a functional fragment or derivative thereof, is administered each time. In other embodiments, a different protein M, or a functional fragment or derivative thereof, is administered each time (e.g., a different modified protein M, as further described below). Without being bound by theory, it is believed that using a different protein M derivative with each administration may limit the effect of inhibitory antibodies against protein M that may arise from re-administration of the same protein. It is also believed that administration of a saturating dose of Protein M or a functional fragment or derivative thereof will overcome any inhibitory antibodies against Protein M and prevent antigen recognition.

[0108] The ability of Protein M or its functional fragments or derivatives to advantageously bind non-specifically to antibodies can be used in other ways in which it is advantageous to inhibit antibodies from binding to antigens, for example when immunosuppression is desired or when excess antibodies are present.

[0109] A further aspect of the present invention relates to a method for treating an autoimmune disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, thereby treating the autoimmune disease.

[0110] The term "autoimmune disease" as used herein refers to any disorder associated with autoimmune reaction.Examples include, but are not limited to, multiple sclerosis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, rheumatoid arthritis, irritable bowel syndrome, uveitis, insulin-dependent diabetes mellitus, hemolytic anemia, rheumatic fever, Goodpasture's syndrome, Guillain-Barre syndrome, psoriasis, thyroiditis, Graves' disease, myasthenia gravis, glomerulonephritis, and autoimmune hepatitis.

[0111] Another aspect of the present invention relates to a method for treating a disorder associated with excess antibodies in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, thereby treating the disorder associated with excess antibodies. As used herein, the term "excess antibody-associated disorder" refers to any disorder in which the cause of the disorder or at least one symptom is due to higher-than-average levels of antibodies in the blood or elsewhere in the body. Examples include, but are not limited to, multiple myeloma, monoclonal gammopathy of undetermined significance (MGUS), and Waldenstrom's macroglobulinemia. The method may also be useful for acutely blocking all antibodies to rapidly stop autoimmune events, such as cytokine release syndrome or acute autoimmune attacks, including sudden-onset severe autoimmune vasculitis, or for preventing damage to transplanted tissues caused by antibody-mediated immune complex formation.

[0112] For any of the methods of the present invention, Protein M or a functional fragment or derivative thereof can be administered to a subject by any route of administration found to be effective. The most appropriate route will depend on the subject being treated and the disorder or condition being treated. In some embodiments, Protein M or a functional fragment or derivative thereof is administered to a subject by a route selected from oral, rectal, transmucosal, intranasal, inhalation (e.g., via aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, intravitreal, intracochlear, transdermal, intraendothelial, intrauterine (or intraembryonic), parenteral (e.g., intravenous, subcutaneous, intradermal, intracranial, intramuscular (including administration to skeletal muscle, diaphragm, and / or cardiac muscle), intrapleural, intracerebral, and intraarticular), topical (e.g., both to the skin and mucosal surfaces (including respiratory tract surfaces), and transdermal administration), intralymphatic, etc., and direct tissue or organ injection (e.g., into the liver, eye, skeletal muscle, cardiac muscle, diaphragm muscle, or brain).

[0113] Protein M or a functional fragment or derivative thereof can be delivered or targeted to any tissue or organ within a subject. In some embodiments, Protein M or a functional fragment or derivative thereof is administered to, for example, skeletal muscle, smooth muscle, heart, diaphragm, respiratory epithelium, liver, kidney, spleen, pancreas, skin, lung, ear, and eye. In some embodiments, Protein M or a functional fragment or derivative thereof is administered to a diseased tissue or organ, such as a tumor.

[0114] In some embodiments, the heterologous agent and protein M or a functional fragment or derivative thereof are administered by the same route. In other embodiments, the heterologous agent and protein M or a functional fragment or derivative thereof are administered by different routes, e.g., protein M or a functional fragment or derivative thereof is administered intravenously and the heterologous agent is administered locally to the target tissue or organ.

[0115] Any of the above methods may further comprise administering to the subject an additional treatment to reduce antibody concentration or inhibit antibody function in the subject. The additional treatment may be any method known in the art, including, but not limited to, plasma exchange, administration of antibody-digesting enzymes such as IdeS or IdeZ, splenectomy, immunosuppressant drugs (e.g., corticosteroids (e.g., prednisone, budesonide, prednisolone), Janus kinase inhibitors (e.g., tofacitinib), calcineurin inhibitors (e.g., cyclosporine, tacrolimus), mTOR inhibitors (e.g., sirolimus, everolimus), IMDH inhibitors (e.g., azathioprine, leflunomide, mycophenolate), or the like. or biologics (e.g., abatacept, adalimumab, anakinra, certolizumab, etanercept, golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, vedolizumab, basiliximab, daclizumab), or treatments designed to inhibit or destroy B cells (e.g., chemotherapy, immunotherapy, radiation therapy). The additional treatment may be administered before, during, and / or after administration of Protein M or a functional fragment or derivative thereof.

[0116] The ability of Protein M or its functional fragments or derivatives to advantageously bind nonspecifically to antibodies can be used in purification methods. While antibody purification often relies on agents that bind to the Fc region of antibodies (e.g., Protein A and Protein G), Protein M binds nonspecifically to the variable region of antibodies. Thus, Protein M can be used to isolate antibody fragments and derivatives that do not contain the Fc region (e.g., single-chain variable fragments) and other molecules incorporating antibody variable regions.

[0117] Thus, one aspect of the present invention relates to a method for isolating a compound comprising an antibody light chain variable region and / or heavy chain variable region from a sample, the method comprising contacting the compound with a modified Mycoplasma protein M or functional fragment thereof of the present invention attached to a solid support, and then eluting the compound from the modified Mycoplasma protein M or functional fragment thereof. In some embodiments, the compound comprising an antibody light chain variable region and / or heavy chain variable region is an antibody or antigen-binding fragment thereof. In some embodiments, the compound comprising an antibody light chain variable region and / or heavy chain variable region is an antibody derivative, an immunoglobulin scaffold, or the like. The modified protein M or functional fragment thereof of the present invention has an advantage over wild-type protein M due to its increased thermal stability. This allows protein M to be reused for multiple purifications and allows the use of elution conditions that would otherwise destabilize wild-type protein M.

[0118] The method can be performed using techniques well known in the field of affinity purification. The solid support can be any material suitable for affinity chromatography or batch purification. Suitable materials include, but are not limited to, agarose, polyacrylamide, dextran, cellulose, polysaccharides, nitrocellulose, silica, alumina, aluminum oxide, titania, titanium oxide, zirconia, styrene, polyvinyl difluoride nylon, copolymers of styrene and divinylbenzene, polymethacrylate esters, derivatized azlactone polymers or copolymers, glass, or cellulose. In some embodiments, the solid support is a resin. In some embodiments, the solid support is a bead or particle. In some embodiments, the solid support is the surface of, for example, a plate, vial, or column.

[0119] The contacting step can be carried out by any suitable method, such as by passing a sample containing the compound over the modified Mycoplasma protein M or functional fragment thereof in a column, or by incubating a composition containing the compound with the modified Mycoplasma protein M or functional fragment thereof in a container or plate well. The contacting step can be carried out for a time sufficient to allow the compound to bind to the modified Mycoplasma protein M or functional fragment thereof. After washing, centrifugation, or other separation of the compound bound to the modified Mycoplasma protein M or functional fragment thereof from other components in the sample, the compound is eluted from the modified Mycoplasma protein M or functional fragment thereof. Elution can be carried out by any method known in the art, such as by changes in ion concentration, temperature, etc. In one embodiment, elution is carried out by a change in pH. The modified Mycoplasma protein M or functional fragment thereof of the present invention is advantageously stable over a wider pH range than wild-type protein M. This allows the modified protein M to remain stable at lower pHs, which allows for elution of the compound.

[0120] In some embodiments, the contacting step is performed in a binding buffer (e.g., neutral pH) and elution is performed using a low pH buffer (e.g., 0.1 M glycine pH 2-3.5 or 0.1 M acetate pH 3.5-4.5) into a neutralization buffer (e.g., a high ionic strength alkaline buffer such as 1 M phosphate or 1 M Tris (pH 8-9)).

[0121] A further aspect of the present invention relates to a modified Mycoplasma protein M or a functional fragment thereof attached to the above-mentioned solid support. The modified Mycoplasma protein M or a functional fragment thereof can be attached to the solid support by any means known in the art, such as by covalent bonding, for example, using a linker molecule.

[0122] The ability of Protein M or a functional fragment or derivative thereof to bind advantageously non-specifically to an antibody may be used in any immunoassay which includes a step of binding to an antibody or a fragment or derivative thereof.

[0123] Accordingly, one aspect of the present invention relates to a method for performing an immunoassay, the method comprising the step of using a modified Mycoplasma protein M of the present invention or a functional fragment thereof to bind to a compound comprising an antibody light chain variable region and / or a heavy chain variable region.

[0124] The modified Mycoplasma protein M or functional fragments thereof can be used in place of any generic or specific antibody binding molecule, e.g., Protein A, Protein G, or secondary antibodies. The modified Mycoplasma protein M or functional fragments thereof may be labeled for, e.g., radioactive, chemiluminescent, or enzymatic detection, as is well known in the art.

[0125] Examples of immunoassays include, but are not limited to, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), enzyme-linked immunosorbent assays (EIAs), sandwich assays, gel diffusion precipitation reactions, immunodiffusion assays, agglutination assays, immunofluorescence assays, fluorescence-activated cell sorting (FACS) assays, immunohistochemistry assays, protein A immunoassays, protein G immunoassays, protein L immunoassays, biotin / avidin assays, biotin / streptavidin assays, immunoelectrophoresis assays, precipitation / flocculation reactions, immunoblots (Western blots; dot / slot blots); immunodiffusion assays; liposome immunoassays, chemiluminescence assays, library screening, expression arrays, immunoprecipitations, competitive binding assays, and immunohistochemistry staining.

[0126] Protein M and its derivatives Protein M or a functional fragment or derivative thereof for use in the present invention may be derived from any mycobacterial species that produces protein M that binds to antibodies. In some embodiments, protein M or a functional fragment or derivative thereof is derived from Mycoplasma genitalium, Mycoplasma pneumoniae, or Mycoplasma penetrans.

[0127] In some embodiments, protein M, or a functional fragment or derivative thereof, can be any protein M sequence described in PCT Publication No. WO2014 / 014897 and U.S. Publication No. 2017 / 0320921, which are incorporated by reference in their entireties. In some embodiments, protein M, or a functional fragment or derivative thereof, is M. genitalium protein M (MG281, SEQ ID NO: 1) or a functional fragment or derivative thereof (e.g., the fragment set forth in SEQ ID NO: 3 or a derivative thereof). In some embodiments, protein M, or a functional fragment or derivative thereof, is M. pneumoniae protein M (MPN400, SEQ ID NO: 23) or a functional fragment or derivative thereof (e.g., the fragment set forth in SEQ ID NO: 24 or a derivative thereof). In some embodiments, protein M or a functional fragment or derivative thereof is a protein M fragment or derivative of a fragment, e.g., a fragment that does not include the transmembrane domain and / or a fragment that does not include the C-terminus, e.g., a functional fragment comprising, consisting essentially of, or consisting of about amino acid residues 17-537, 37-556, 37-482, 37-468, 37-442, 74-468, 74-479, 74-482, 74-468, 74-442, or 74-556 of M. genitalium protein M (SEQ ID NO: 1), or the corresponding residues from another protein M. The term "about" as applied to each end of the recited fragment means that one or both of the terminal residues may differ to a small extent, e.g., by about 5, 4, 3, or 2 amino acids on either side of the recited residue. The corresponding residues from another protein M can be readily determined by one of skill in the art by performing sequence alignments between M. genitalium protein M and other proteins M. For example, Figure 27 shows a sequence alignment of wild-type M. genitalium protein M amino acids 74-479 (SEQ ID NO:3) and the corresponding fragment of M. pneumoniae protein M (SEQ ID NO:24).In some embodiments, Protein M or a functional fragment or derivative thereof comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 2, which is a soluble form of Protein M (amino acid residues 37 to 556 of SEQ ID NO: 1) with an N-terminal 6-His tag followed by a thrombin cleavage site.

[0128] As used herein, the term "derivative" refers to a polypeptide that differs from naturally occurring protein M or a functional fragment of protein M by minor modifications to the naturally occurring polypeptide, but that largely maintains the biological activity of protein M. Minor modifications include, but are not limited to, alterations of one or several amino acid side chains, alterations of one or several amino acids (including deletions, insertions, and / or substitutions), alterations in the stereochemistry of one or several atoms (e.g., D-amino acids), and minor derivatizations, including, but not limited to, methylation, glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation, and addition of glycosylphosphatidylinositol. As used herein, the term "substantially maintain" refers to a fragment, derivative, or other variant of a polypeptide that maintains at least about 20%, e.g., about 30%, 40%, 50%, or more of the activity (e.g., antibody binding) of the naturally occurring polypeptide. In some embodiments, a derivative of protein M or a functional fragment of protein M comprises mutations (deletions, insertions, and / or substitutions in any combination) of 20 or fewer amino acid residues, e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 or fewer mutations. In some embodiments, a derivative of protein M comprises an amino acid sequence that is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identical to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 of M. pneumoniae protein M or the wild-type sequence of another mycoplasmal protein M or functional fragment thereof.

[0129] In some embodiments, Protein M or a functional fragment or derivative thereof can be modified for in vivo use by the addition of a blocking agent at the amino and / or carboxyl terminus to promote in vivo survival of the relevant polypeptide. This can be useful in situations where the peptide termini are prone to degradation by proteases. Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and / or carboxyl terminal residues of the administered protein. This can be done chemically during protein synthesis or by recombinant DNA technology using methods familiar to those of ordinary skill in the art. Alternatively, a blocking agent, such as pyroglutamic acid or other molecules known in the art, can be attached to the amino and / or carboxyl terminal residues, or the amino group at the amino terminus or the carboxyl group at the carboxyl terminus can be replaced with a different moiety. Similarly, proteins can be covalently or noncovalently linked to a pharmaceutically acceptable "carrier" protein prior to administration.

[0130] In one aspect of the present invention, a protein M derivative is a modified Mycoplasma protein M or a functional fragment thereof that contains mutations that increase or at least maintain the thermal stability of protein M. These modified protein M derivatives are highly suitable for use in in vivo methods and other methods that require high temperatures (e.g., about 37°C) at which wild-type protein M can denature.

[0131] In some embodiments, the protein M derivative is a modified Mycoplasma protein M or a functional fragment thereof having one or more amino acid mutations that increase or maintain the thermal stability of the Mycoplasma protein M or a functional fragment thereof compared to wild-type Mycoplasma protein M or a functional fragment thereof. In some embodiments, the modified protein M or functional fragment thereof has a melting temperature (Tm) that is increased by at least 0.5°C, e.g., 0.5°C, 1.0°C, 1.5°C, 2.0°C, 2.5°C, 3.0°C, 3.5°C, 4.0°C, 4.5°C, 5.0°C, 5.5°C, 6.0°C, 6.5°C, 7.0°C, 7.5°C, 8.0°C, 8.5°C, 9.0°C, 9.5°C, 10.0°C, 11.0°C, 12.0°C, 13.0°C, 14.0°C, 15.0°C, 16.0°C, 17.0°C, 18.0°C, 19.0°C, 20.0°C, or more, compared to the Tm of wild-type protein M or functional fragment thereof. In some embodiments, the modified protein M or functional fragment thereof has a maintained Tm (i.e., within 0.5°C) compared to the Tm of wild-type protein M or functional fragment thereof. Tm can be measured by differential scanning fluoroscopy or any other suitable technique. The Tm of wild-type Mycoplasma genitalium protein M is about 41.9°C, and the Tm of wild-type Mycoplasma pneumoniae protein M is about 44.1°C.

[0132] In some embodiments, the modified Mycoplasma protein M or functional fragment thereof may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more mutations. In some embodiments, the modified Mycoplasma protein M or functional fragment thereof may have 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer mutations.

[0133] In some embodiments, the modified Mycoplasma protein M or functional fragment thereof is derived from protein M of Mycoplasma genitalium or Mycoplasma pneumoniae.

[0134] In some embodiments, the modified Mycoplasma protein M or functional fragment thereof is a fragment of M. genitalium protein M from about residue 74 (e.g., residues 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79) to about residue 479 (e.g., residues 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484) (SEQ ID NO: 3) or the corresponding residues of M. pneumoniae protein M (SEQ ID NO: 24).

[0135] In some embodiments, the one or more mutations are located in portions of protein M known to affect thermostability. In some embodiments, the one or more mutations are not located in proteins of protein M known to have other roles in the biological activity of protein M. In one embodiment, the one or more mutations are located in residues within 5 Å of the antibody binding site of protein M of M. genitalium protein M (SEQ ID NO: 1) (i.e., residues 95, 99, 102, 103, 105, 106, 107, 109, 110, 114, 116, 117, 118, 119, 120, 144, 158, 160, 161, 162, 163, 177, 178, 179, 180, 181, 186, 187, 188, 191, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371 , 338, 340, 341, 345, 381, 384, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 426, 427, 429, 436, 438, 439, 440, 441, 442, 444, 445, 446, 447, 448, 449, 452, 453, 455, 456, 457, 462, 466) or in the corresponding residues of M. pneumoniae protein M (SEQ ID NO: 23). In one embodiment, the one or more mutations are at residues within 5 Å of the antibody binding site of M. pneumoniae protein M (SEQ ID NO: 23) of protein M (i.e., residues 100, 104, 107, 108, 110, 111, 112, 114, 115, 119, 121, 122, 123, 124, 125, 149, 163, 165, 166, 167, 168, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, , 184, 185, 186, 192, 193, 196, 337, 338, 354, 356, 357, 399, 402, 404, 405, 406, 407, 408, 409, 410, 411, 412, 442, 443, 445, 454, 455, 456, 457, 458, 460, 461, 462, 463, 464, 465, 468, 469, 472, 473, 478). In one embodiment, the one or more mutations are not in any of residues 469-479 of M. genitalium protein M (SEQ ID NO: 1) or the corresponding residues of M. pneumoniae protein M (SEQ ID NO: 23).

[0136] The inventors have used computational analysis to identify residues within protein M that, when mutated, are predicted to increase or maintain the Tm of the protein. As a result, in some embodiments, one or more mutations are made to residues 78, 81, 83, 84, 85, 89, 90, 91, 92, 93, 94, 96, 97, 100, 101, 108, 111, 112, 113, 122, 123, 125, 126, 127, 128, 130, 131, 133, 134, 136, 137, 139, 141, 142, 146, 147, 148, 149, 150, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 204, 205, 206, 207, 208, 209, 210, 155, 156, 164, 167, 170, 175, 176, 184, 185, 189, 192, 193, 196, 198, 201, 202, 204, 205, 206, 207, 209, 211, 215, 218, 220, 224, 225, 226, 227, 231, 232, 234, 235, 236, 237, 239, 241, 243, 244, 245, 246, 247, 249, 250, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 64, 269, 270, 272, 274, 275, 276, 279, 282, 284, 286, 287, 288, 291, 297, 299, 300, 302, 303, 304, 305, 307, 308, 309, 310, 311, 313, 317, 318, 319, 320, 322, 326, 327, 329, 331, 332, 333, 335, 337, 342, 343, 347, 348, 351, 354, 355, 357, 358, 359, 360, 361, 362 2, 363, 367, 369, 370, 371, 372, 373, 374, 375, 378, 385, 399, 400, 401, 402, 405, 406, 407, 408, 409, 411, 413, 414, 417, 418, 419, 424, 428, 434, 435, 443, 450, 459, 460, 463, 464, 465, 468, or any combination thereof, or at the corresponding residues of M. pneumoniae protein M (SEQ ID NO: 23). In some embodiments, the one or more mutations are a mutation listed in Table 4 or any combination thereof. In some embodiments, the one or more mutations areResidues 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 101, 102, 103, 105, 106, 109, 113, 116, 117, 118, 120, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233 8, 159, 160, 161, 162, 164, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 187, 188, 189, 190, 191, 194, 195, 197, 198, 199, 200, 201, 202 , 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 65, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296 6, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327 , 328, 329, 330, 331, 332, 333, 334, 335, 336, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 355, 358, 359, 360, 361, 362, 363,364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 400, 401, 403, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422 , 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 444, 446, 447, 448, 449, 450, 451, 452, 453, 459, 466, 467, 470, 471, 474, 475, 476, 477, 479, 480, 481, 482, 483, 484, or any combination thereof.

[0137] In some embodiments, the one or more mutations are at residues 83, 90, 92, 94, 137, 142, 147, 150, 156, 184, 196, 198, 205, 211, 215, 225, 231, 232, 234, 235, 236, 237, 239, 243, 245, 250, 255, 256, 259, 264, 272, 274, 275, 276, 277, 278, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 339, 343, 345, 350, 355, 356, 359, 364, 365, 366, 367, 368, 369, 370, 371, 6, 279, 282, 297, 300, 302, 310, 320, 326, 331, 332, 335, 342, 343, 347, 348, 355, 357, 361, 371, 374, 378, 385, 401, 402, 409, 413, 424, 460, 463, 464, 468, or any combination thereof, or at the corresponding residues in M. pneumoniae protein M (SEQ ID NO: 23). In some embodiments, the one or more mutations are those listed in Table 5 or any combination thereof.

[0138] We prepared and tested a large number of mutations from the predicted residue list, either alone or in combination with a high success rate of increasing thermostability (stabilizing mutations) or at least maintaining thermostability (neutral mutations). See Figure 23, which shows that 79% of the point mutations tested were stabilizing or neutral. The data further show that combinations of point mutations that increase Tm tend to yield modified protein M with even higher Tm (see Figure 15A).

[0139] Thus, in some embodiments, the one or more mutations are at residues shown to increase Tm, either alone or in combination with other mutations, for example, the one or more mutations are at residues 150, 196, 198, 201, 205, 224, 232, 237, 274, 282, 342, 355, 373, 400, 402, 407, 409, 413, 135, or any combination thereof, of M. genitalium protein M (SEQ ID NO: 1), or the corresponding residues of M. pneumoniae protein M (SEQ ID NO: 23).

[0140] In some embodiments, the one or more mutations are in a residue selected from the following residues or combinations of residues: M. genitalium protein M (SEQ ID NO: 1), a) 237(MG1); b) 232(MG8); c) 282(MG13); d) 150, 196, 198, 400, 402, 407, 409 (MG15); e) 413, 435(MG21); f) 373, 400(MG22); g)402, 407, 409, 413 (MG23); h)342(MG24); i) 150, 196, 198, 232, 237, 282, 342, 373, 400, 402, 407, 409, 413, 435 (MG27); j)274(MG28); k)150, 196, 198, 232, 237, 342, 400, 402, 407, 409 (MG29); l) 373, 413, 435 (MG31) m)205(MG33); n)355(MG38, MG40); o) 150, 196, 198, 342, 373, 400, 402, 407, 409 (MG43); p)150, 196, 198, 232, 237, 342, 373, 400, 402, 407, 409 (MG44); q)201, 224(MG45); r)150, 196, 198, 201, 224, 232, 237, 342, 400, 402, 407, 409 (MG46); s)150, 196, 198, 232, 237, 342, 390, 400, 402, 407, 409, 444 (MG47); t) 150, 196, 198, 201, 205, 224, 232, 237, 274, 342, 355, 400, 402, 407, 409 (MG48); or u)150, 196, 198, 232, 237, 342, 391, 400, 402, 407, 409 (MG49) or the corresponding residues of M. pneumoniae protein M (SEQ ID NO: 23).

[0141] In some embodiments, the one or more mutations are selected from the following: M. genitalium protein M (SEQ ID NO: 1), a) F237T(MG1); b) S232Q(MG8); c) Q282D(MG13); d)S150E, S196R, S198P, V400I, N402I, K407P, S409V(MG15); e) L413I, T435I(MG21); f) V373I, V400I(MG22); g)N402L, K407P, S409V, L413I(MG23); h)A342V(MG24); i) S150E, S196R, S198P, S232Q, F237T, Q282D, A342V, V373I, V400I, N402I, K407P, S409V, L413I, T435I (MG27); j) N274D(MG28); k)S150E, S196R, S198P, S232Q, F237T, A342V, V400I, N402I, K407P, S409V(MG29); l) V373I, L413I, T435I (MG31) m)A205P(MG33); n)T355D(MG38); o)T355P(MG40); p)S150E, S196R, S198P, A342V, V373I, V400I, N402I, K407P, S409V(MG43); q) 150, 196, 198, 232, 237, 342, 373, 400, 402, 407, 409 (MG44); r)S201C, A224C(MG45); s)S150E, S196R, S198P, S201C, A224C, S232Q, F237T, A342V, V400I, N402I, K407P, S409V(MG46) t)S150E, S196R, S198P, S232Q, F237T, A342V, F390E, V400I, N402I, K407P, S409V Y444K(MG47); u) S150E, S196R, S198P, S201C, A205P, A224C, S232Q, F237T, N274D, A342V, T355P, V400I, N402I, K407P, S409V (MG48); or v)S150E, S196R, S198P, S232Q, F237T, A342V, A391P, V400I, N402I, K407P, S409V(MG49) or the corresponding residues of M. pneumoniae protein M (SEQ ID NO: 23).

[0142] In some embodiments, the one or more mutations are at residues shown to maintain Tm, either alone or in combination with other mutations, e.g., the one or more mutations are at residues 147, 150, 156, 225, 232, 245, 272, 276, 277, 279, 300, 310, 355, 378, 468, or any combination thereof, of M. genitalium protein M (SEQ ID NO: 1), or the corresponding residues of M. pneumoniae protein M (SEQ ID NO: 23).

[0143] In some embodiments, the one or more mutations are in a residue selected from the following residues or combinations of residues: M. genitalium protein M (SEQ ID NO: 1), a) 468(MG2); b) 150(MG4); c) 147(MG5); d) 272(MG10); e) 355(MG12); f) 276, 277, 279 (MG17); g) 300(MG18); h)378(MG20); i) 156(MG32); j)232(MG34); k)245(MG35); l)276(MG36) m)225(MG41); or n)310(MG42) or the corresponding residues of M. pneumoniae protein M (SEQ ID NO: 23).

[0144] In some embodiments, the one or more mutations are selected from the following: M. genitalium protein M (SEQ ID NO: 1), a) R468Q(MG2); b) S150E(MG4); c) H147F(MG5); d) S272G(MG10); e) T355G(MG12); f)S276E, Q277L, N279R(MG17); g) N300Q(MG18); h) N378Y(MG20); i) S156K (MG32); j)S232L(MG34); k)A245Q(MG35); l) S276D(MG36) m)K225P(MG41); or n) V310E (MG42) or the corresponding residues of M. pneumoniae protein M (SEQ ID NO: 23).

[0145] In some embodiments, the one or more mutations are at residues 155, 203, 243, 248, and 358 of M. pneumoniae protein M (SEQ ID NO: 23).

[0146] In some embodiments, the one or more mutations are A155E, K203R, H243T, V248Q, and A358V of M. pneumoniae protein M (SEQ ID NO: 23).

[0147] The modified Mycoplasma protein M or functional fragment thereof may contain additional modifications other than mutations within the amino acid sequence. In some embodiments, one or more glycosylation sites within the protein M sequence are removed, for example, one, two, or three glycosylation sites. Three N-glycosylation sites are predicted in M. genitalium protein M based on both sequence and structural analysis using the NGlycPred server. These include N177, N213, and N274. Two O-glycosylation sites are predicted in M. genitalium protein M based on structural analysis using the NetOGlyc 4.0 server. These include T110 and T206. Suitable mutations include, but are not limited to, N177D, T215Y, N274D, S112I, and T206Y, in any combination. In some embodiments, one or more glycosylation sites are added to the modified Mycoplasma protein M or functional fragment thereof. Altering the glycosylation pattern can add to the thermal stability of the protein and / or alter the immunogenicity of the protein by blocking antibody recognition.

[0148] For expression and purification purposes, modified Mycoplasma protein M or functional fragments may contain a secretory peptide, e.g., at the N-terminus, so that the expressed protein can be secreted from the cells in which it is expressed and recovered from the culture medium. Suitable secretory peptides include, but are not limited to, those derived from human serum albumin, interleukin-2, CD5, immunoglobulin kappa light chain, trypsinogen, or prolactin (for mammalian cells), and Sec or Tat (for bacterial cells). The secretory peptide may or may not be removed from protein M before use in the methods of the invention.

[0149] In some embodiments, the modified Mycoplasma protein M or functional fragment may contain one or more additional mutations that alter one or more biological functions or physical characteristics of the protein. In some embodiments, the modified Mycoplasma protein M or functional fragment may contain one or more additional mutations that alter the affinity of the protein for an antibody. The inventors have used computational analysis to identify residues within protein M that, when mutated, are predicted to increase the affinity of the protein for an antibody. As a result, in some embodiments, the one or more mutations are at residues 95, 102, 103, 106, 107, 114, 116, 160, 161, 162, 163, 181, 186, 321, 381, 384, 389, 390, 391, 396, 397, 426, 429, 436, 438, 439, 441, 442, 447, 448, 449, 452, 453, 455, 456, 462, or 466 of M. genitalium protein M (SEQ ID NO: 1), or any combination thereof, or the corresponding residues of M. pneumoniae protein M (SEQ ID NO: 23). In some embodiments, the one or more mutations are those listed in Table 6, or any combination thereof. In some embodiments, the one or more mutations are at residues 100, 104, 107, 108, 110, 111, 112, 114, 115, 119, 121, 122, 123, 124, 125, 149, 163, 165, 166, 167, 168, 182, 183, 184, 185, 186, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 2 , 196, 337, 338, 354, 356, 357, 399, 402, 404, 405, 406, 407, 408, 409, 410, 411, 412, 442, 443, 445, 454, 455, 456, 457, 458, 460, 461, 462, 463, 464, 465, 468, 469, 472, 473, 478, or any combination thereof.

[0150] In some embodiments, the modified Mycoplasma protein M or functional fragment may contain one or more additional mutations that alter the protein's affinity for antibodies by modifying its pH sensitivity. Using computational analysis, the inventors have identified residues within protein M that, when mutated, are predicted to increase affinity for antibodies by modifying pH sensitivity. These mutants may be particularly useful for antibody isolation due to their ability to use pH changes for elution. Thus, in some embodiments, the one or more mutations are at residues 95, 103, 116, 186, 321, 389, 429, 442, or 466 of M. genitalium protein M (SEQ ID NO: 1), or any combination thereof, or the corresponding residues of M. pneumoniae protein M (SEQ ID NO: 23). In some embodiments, the one or more mutations are those listed in Table 7 or any combination thereof.

[0151] In some embodiments, the modified Mycoplasma protein M or functional fragment may contain one or more additional mutations that reduce or eliminate affinity for the antibody. Examples include, but are not limited to, mutations at residues 390 and 444 of M. genitalium protein M (SEQ ID NO: 1), e.g., 390E and Y444K, or the corresponding residues of M. pneumoniae protein M (SEQ ID NO: 23).

[0152] Protein M proteins of the present invention may be produced and characterized by methods well known in the art and described herein, such as by recombinant expression.

[0153] A further aspect of the present invention provides isolated polynucleotides encoding protein M or a functional fragment or derivative thereof of the present invention and expression cassettes for producing protein M or a functional fragment or derivative thereof.

[0154] The polynucleotide may be operably linked to regulatory elements to facilitate expression of the protein. In some embodiments, the polynucleotide is operably linked to a promoter. The promoter may be a bacterial promoter (e.g., operable in E. coli) or a mammalian promoter (e.g., a human promoter).

[0155] In some embodiments, the polynucleotide may be codon-optimized to enhance protein expression in a host cell. In one embodiment, the polynucleotide is codon-optimized for expression in bacteria, such as E. coli. In another embodiment, the polynucleotide is codon-optimized for expression in mammalian cells, such as human cells. One example is the sequence of SEQ ID NO: 26, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto, that has been codon-optimized for expression in human cells. In a further embodiment, the polynucleotide is codon-optimized for expression in both bacteria, such as E. coli, and mammalian cells, such as human cells. One example is the sequence of SEQ ID NO: 25, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto, that has been codon-optimized for expression in both E. coli and human cells.

[0156] Another aspect of the present invention is a vector, e.g., an expression vector, comprising a polynucleotide of the present invention. The vector may be any type of vector known in the art, including, but not limited to, a plasmid vector and a viral vector. The vector may be, for example, a bacterial vector (e.g., an E. coli vector) or a mammalian cell vector (e.g., a human cell vector).

[0157] A further aspect of the present invention relates to cells (e.g., isolated cells, transformed cells, recombinant cells, etc.) comprising the polynucleotides and / or vectors of the present invention. Accordingly, various embodiments of the present invention relate to recombinant host cells comprising the vectors (e.g., expression cassettes). Such cells may be isolated cells. In some embodiments, the polynucleotides are stably integrated into the genome of the cells. In some embodiments, the cells may be bacterial cells, such as E. coli, or mammalian cells, such as human cells.

[0158] A further aspect of the present invention relates to a kit comprising the modified Mycoplasma protein M or functional fragment thereof, polynucleotide, vector, and / or transformed cell of the present invention. The kit may include additional reagents for performing one of the methods described herein. The reagents may be contained in suitable packaging or containers. The additional reagents may include, but are not limited to, buffers, labels, enzymes, detection reagents, etc.

[0159] When a kit is provided, different components may be packaged in separate containers and mixed immediately before use. Such separate packaging of components may allow for long-term storage without losing the functionality of the active ingredients. The kit may also be provided with instruction materials. The instructions may be printed on paper or another substrate and / or provided as an electronically readable medium.

[0160] Different drugs As described above, the heterologous agent may be one for which neutralizing antibodies exist in the subject before administration of the heterologous agent, or one for which neutralizing antibodies may be produced upon administration to the subject. In some embodiments, the heterologous agent may be a nucleic acid delivery vector (e.g., a viral vector or a non-viral vector), a gene editing complex (e.g., a CRISPR complex), a protein, or a nucleic acid.

[0161] Any nucleic acid sequence(s) of interest can be delivered in the nucleic acid delivery vectors of the invention. Nucleic acids of interest include nucleic acids encoding polypeptides, including therapeutic (e.g., for pharmaceutical or veterinary uses), immunogenic (e.g., for vaccines), or diagnostic polypeptides.

[0162] Therapeutic polypeptides include, but are not limited to, cystic fibrosis transmembrane conductance regulator (CFTR), dystrophin (mini- and micro-dystrophin) (e.g., Vincent et al., (1993) Nature Genetics 5:130; U.S. Patent Publication No. 2003 / 017131; WO / 2008 / 088895; Wang et al., Proc. Natl. Acad. Sci. USA 97:13714-13719 (2000);and Gregorevic et al., Mol. Ther. 16:657-64 (2008)), myostatin propeptide, follistatin, activin type II soluble receptor, IGF-1, anti-inflammatory polypeptides such as IkappaB dominant mutants, sarcospan, utrophin (Tinsley et al., (1996) Nature 384:349), mini-utrophin, coagulation factors (e.g., factor VIII, factor IX, factor X, etc.), erythropoietin, angiostatin, endostatin, catalase, tyrosine hydroxylase, superoxide dismutase, leptin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β-globin, α-globin, spectrin, α1-antitrypsin, adenosine deaminase, hypoxanthine guanine phosphoribosyltransferase, β-glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase A, branched-chain ketoacid dehydrogenase, RP65 protein, cytokines (e.g., α-interferon, β-interferon, interferon-γ, interleukin-2, interleukin-4, granulocyte-macrophage colony-stimulating factor, lymphotoxin, etc. ), peptide growth factors, neurotrophic factors and hormones (e.g., somatotropin, insulin, insulin-like growth factors 1 and 2, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, nerve growth factor, neurotrophic factor-3 and -4, brain-derived neurotrophic factor, bone morphogenetic proteins [including RANKL and VEGF], glial-derived growth factor, transforming growth factor-α and -β, etc.), lysosomal acid α-glucosidase, α-galactosidase A, receptors (e.g., tumor necrosis growth factor α soluble receptor), S100A1, parvalbumin, adenylyl cyclase type 6, molecules that cause G protein-coupled receptor kinase type 2 knockdown, e.g., truncated constitutively active bARKct, anti-inflammatory factors such as IRAP, anti-myostatin protein, aspartoacylase, and monoclonal antibodies (including single-chain monoclonal antibodies;An exemplary Mab is the Herceptin® Mab. Other exemplary heterologous nucleic acid sequences encode suicide gene products (e.g., thymidine kinase, cytosine deaminase, diphtheria toxin, and tumor necrosis factor), proteins that confer resistance to drugs used in cancer therapy, tumor suppressor gene products (e.g., p53, Rb, Wt-1), TRAIL, FAS-ligand, and any other polypeptides that have a therapeutic effect in a subject in need thereof. Parvoviral vectors can also be used to deliver monoclonal antibodies and antibody fragments, for example, antibodies or antibody fragments directed against myostatin (see, e.g., Fang et al., Nature Biotechnol. 23:584-590 (2005)).

[0163] Nucleic acid sequences encoding polypeptides include those that encode reporter polypeptides (e.g., enzymes). Reporter polypeptides are known in the art and include, but are not limited to, green fluorescent protein, β-galactosidase, alkaline phosphatase, luciferase, and chloramphenicol acetyltransferase genes.

[0164] Alternatively, in certain embodiments of the invention, the nucleic acid may encode a functional nucleic acid, i.e., a nucleic acid that functions without being translated into a protein, such as an antisense nucleic acid, a ribozyme (e.g., as described in U.S. Pat. No. 5,877,022), an RNA that causes spliceosome-mediated trans-splicing (see Puttaraju et al., (1999) Nature Biotech. 17:246; U.S. Pat. No. 6,013,487; U.S. Pat. No. 6,083,702), an interfering RNA (RNAi) including an siRNA, shRNA, or miRNA that mediates gene silencing (see Sharp et al., (2000) Science 287:2431), and other non-translated RNA, such as a "guide" RNA (Gorman et al., (1998) Proc. Nat. Acad. Sci. USA 95:4929; U.S. Pat. No. 5,869,248 (Yuan et al.)).Exemplary non-coding RNAs include RNAi against multidrug resistance (MDR) gene products (e.g., for administration to the heart to treat and / or prevent tumors and / or to prevent chemotherapy damage), RNAi against myostatin (e.g., for Duchenne muscular dystrophy), RNAi against VEGF (e.g., for treating and / or preventing tumors), RNAi against phospholamban (e.g., for treating cardiovascular disease, see, e.g., Andino et al., J. Gene Med. 10:132-142 (2008) and Li et al., Acta Pharmacol Sin. 26:51-55 (2005)); phospholamban inhibitors or dominant-negative molecules, such as phospholamban S16E (e.g., for treating cardiovascular disease, see, e.g., Hoshijima et al. al. Nat. Med. 8:864-871 (2002)), RNAi against adenosine kinase (e.g., for epilepsy), RNAi against sarcoglycans [e.g., α, β, γ], RNAi against myostatin, myostatin propeptide, follistatin, or activin type II soluble receptors, RNAi against anti-inflammatory polypeptides, e.g., IkappaB dominant mutants, and RNAi directed against pathogenic organisms and viruses (e.g., hepatitis B virus, human immunodeficiency virus, CMV, herpes simplex virus, human papilloma virus, etc.).

[0165] Alternatively, in certain embodiments of the invention, the nucleic acid may encode protein phosphatase inhibitor I (I-1), serca2a, a zinc finger protein that regulates the phospholamban gene, Barkct, β2-adrenergic receptor, β2-adrenergic receptor kinase (BARK), phosphoinositide-3 kinase (PI3 kinase), a molecule that causes G protein-coupled receptor kinase type 2 knockdown, such as truncated constitutively active bARKct; calsarcin, RNAi against phospholamban; phospholamban inhibitory or dominant-negative molecules, such as phospholamban S16E, enos, inos, or bone morphogenetic proteins (including BNP2, 7, etc., RANKL and / or VEGF).

[0166] The nucleic acid delivery vector may contain a nucleic acid that shares homology with and recombines with a locus on a host chromosome. This technique can be used, for example, to correct a genetic defect in the host cell.

[0167] The present invention also provides nucleic acid delivery vectors expressing immunogenic polypeptides, e.g., for vaccination. The nucleic acid may encode any immunogen of interest known in the art, including, but not limited to, immunogens derived from human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), influenza virus, HIV or SIV gag proteins, tumor antigens, cancer antigens, bacterial antigens, viral antigens, etc.

[0168] The use of parvoviruses as vaccine vectors is known in the art (see, e.g., Miyamura et al., (1994) Proc. Nat. Acad. Sci USA 91:8507; U.S. Patent No. 5,916,563 (Young et al.); U.S. Patent No. 5,905,040 (Mazzara et al.); U.S. Patent No. 5,882,652; U.S. Patent No. 5,863,541 (Samulski et al.)). Antigens can be present within the parvovirus capsid. Alternatively, antigens can be expressed by nucleic acids introduced into a recombinant vector genome. Any immunogen of interest described herein and / or known in the art can be provided by a nucleic acid delivery vector.

[0169] The immunogenic polypeptide can be any polypeptide suitable for eliciting an immune response and / or protecting a subject against infection and / or disease, including, but not limited to, microbial, bacterial, protozoan, parasitic, fungal, and / or viral infections and diseases. For example, the immunogenic polypeptide can be an orthomyxovirus immunogen (e.g., an influenza virus immunogen, such as an influenza virus hemagglutinin (HA) surface protein or influenza virus nucleoprotein, or an equine influenza virus immunogen) or a lentivirus immunogen (e.g., an equine infectious anemia virus immunogen, a simian immunodeficiency virus (SIV) immunogen, or a human immunodeficiency virus (HIV) immunogen, such as an HIV or SIV envelope GP160 protein, an HIV or SIV matrix / capsid protein, and an HIV or SIV gag, pol, and env gene product). The immunogenic polypeptide can also be an arenavirus immunogen (e.g., a Lassa virus immunogen, such as a Lassa virus nucleocapsid protein and a Lassa fever envelope glycoprotein), a poxvirus immunogen (e.g., a vaccinia virus immunogen, such as a vaccinia L1 or L8 gene product), a flavivirus immunogen (e.g., a yellow fever virus immunogen or a Japanese encephalitis virus immunogen), a filovirus immunogen (e.g., an Ebola virus immunogen, or a Marburg virus immunogen, such as the NP and GP gene products), a bunyavirus immunogen (e.g., an RVFV, CCHF, and / or SFS virus immunogen), or a coronavirus immunogen (e.g., an infectious human coronavirus immunogen, such as a human coronavirus envelope glycoprotein, or a porcine transmissible gastroenteritis virus immunogen, or an avian infectious bronchitis virus immunogen). The immunogenic polypeptide can also be a polio immunogen, a herpes immunogen (e.g., a CMV, EBV, HSV immunogen), a mumps immunogen, a measles immunogen, a rubella immunogen, a diphtheria toxin or other diphtheria immunogen, a pertussis antigen, a hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C, etc.) immunogen, and / or any other vaccine immunogen now known in the art or later identified as an immunogen.

[0170] Alternatively, the immunogenic polypeptide can be any tumor or cancer cell antigen. The tumor or cancer antigen may be expressed on the surface of cancer cells. Exemplary cancer and tumor cell antigens are described in SA Rosenberg (Immunity 10:281 (1991)).Other exemplary cancer and tumor antigens include, but are not limited to, BRCA1 gene product, BRCA2 gene product, gp100, tyrosinase, GAGE-1 / 2, BAGE, RAGE, LAGE, NY-ESO-1, CDK-4, β-catenin, MUM-1, caspase-8, KIAA0205, HPVE, SART-1, PRAME, p15, melanoma tumor antigen (Kawakami et al., (1994) Proc. Natl. Acad. Sci. USA 91:3515; Kawakami et al., (1994) J. Exp. Med., 180:347; Kawakami et al., (1994) Cancer Res. 54:3124), MART-1, gp100 MAGE-1, MAGE-2, MAGE-3, CEA, TRP-1, TRP-2, P-15, tyrosinase (Brichard et al., (1993) J. Exp. Med. 178:489); HER-2 / neu gene product (U.S. Patent No. 4,968,603), CA125, LK26, FB5 (endosialin), TAG72, AFP, CA19-9, NSE, DU-PAN-2, CA50, SPan-1, CA72-4, HCG, STN (sialyl Tn antigen), c-erbB-2 protein, PSA, L-CanAg, estrogen receptor, milk fat globulin, p53 tumor suppressor protein (Levine, (1993) Ann. Rev. Biochem. 62:623); mucin antigen (International Patent Publication No. WO 90 / 05142); telomerase prostatic acid phosphatase; papilloma virus antigen; and / or antigens now known or later discovered to be associated with the following cancers: melanoma, adenocarcinoma, thymoma, lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's lymphoma), sarcoma, lung cancer, liver cancer, colon cancer, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, brain tumor, and any other cancer or malignant condition now known or later identified (see, e.g., Rosenberg, (1996) Ann. Rev. Med. 47:481-91).

[0171] It will be understood by those skilled in the art that the nucleic acid(s) of interest can be operably associated with appropriate control sequences. For example, a heterologous nucleic acid can be operably associated with expression control elements, such as transcriptional / translational control signals, origins of replication, polyadenylation signals, internal ribosome entry sites (IRES), promoters, and / or enhancers.

[0172] Those skilled in the art will understand that various promoter / enhancer elements can be used depending on the desired level and tissue-specific expression. The promoter / enhancer can be constitutive or inducible depending on the desired expression pattern. The promoter / enhancer can be native or foreign, and can be a natural or synthetic sequence. By foreign, it is meant that the transcription initiation region is not found in the wild-type host into which it is introduced.

[0173] In certain embodiments, the promoter / enhancer element can be native to the target cell or subject to be treated. In representative embodiments, the promoter / enhancer element can be native to the heterologous nucleic acid sequence. The promoter / enhancer element is generally selected to function in the intended target cell(s). Furthermore, in certain embodiments, the promoter / enhancer element is a mammalian promoter / enhancer element. The promoter / enhancer element can be constitutive or inducible.

[0174] Inducible expression control elements are typically advantageous in applications where it is desirable to provide regulation over the expression of a nucleic acid sequence(s). Inducible promoter / enhancer elements for gene delivery can be tissue-specific or preferred promoter / enhancer elements, including muscle-specific or preferred (including cardiac, skeletal, and / or smooth muscle-specific or preferred), neural tissue-specific or preferred (including brain-specific or preferred), eye-specific or preferred (including retina-specific and cornea-specific), liver-specific or preferred, bone marrow-specific or preferred, pancreas-specific or preferred, spleen-specific or preferred, and lung-specific or preferred promoter / enhancer elements. Other inducible promoter / enhancer elements include hormone-inducible and metal-inducible elements. Exemplary inducible promoter / enhancer elements include, but are not limited to, the Tet on / off element, the RU486-inducible promoter, the ecdysone-inducible promoter, the rapamycin-inducible promoter, and the metallothionein promoter.

[0175] In embodiments in which the nucleic acid sequence(s) are transcribed and then translated in the target cell, specific initiation signals are generally included for efficient translation of the inserted protein-coding sequence. These exogenous transcriptional control sequences may include the ATG initiation codon and adjacent sequences, and may be of a variety of origins, both natural and synthetic.

[0176] Nucleic acid delivery vectors provide a means for delivering nucleic acids to a wide range of cells, including dividing and non-dividing cells. Nucleic acid delivery vectors can be used to deliver a nucleic acid of interest to cells in vitro, for example, for ex vivo gene therapy. Nucleic acid delivery vectors are further useful in methods for delivering nucleic acids to a subject in need thereof, for example, to express an immunogenic or therapeutic polypeptide or functional RNA. In this manner, a polypeptide or functional RNA can be produced in vivo in the subject. The subject may need the polypeptide because it is deficient in that polypeptide. Furthermore, the method can be carried out because producing a polypeptide or functional RNA in the subject may have some beneficial effect.

[0177] Nucleic acid delivery vectors can also be used to produce a polypeptide or functional RNA of interest in a subject (e.g., using the subject as a bioreactor to produce a polypeptide or to observe the effect of a functional nucleic acid on the subject, e.g., in conjunction with screening methods).

[0178] In general, the nucleic acid delivery vector of the present invention can be used to deliver nucleic acids encoding a polypeptide or functional nucleic acid to treat and / or prevent any condition in which it is beneficial to deliver a therapeutic polypeptide or functional nucleic acid. Exemplary conditions include, but are not limited to: cystic fibrosis (cystic fibrosis transmembrane conductance regulator) and other diseases of the lung, hemophilia A (factor VIII), hemophilia B (factor IX), thalassemia (β-globin), anemia (erythropoietin) and other blood disorders, Alzheimer's disease (GDF; neprilysin), multiple sclerosis (β-interferon), Parkinson's disease (glial cell line-derived neurotrophic factor [GDNF]), Huntington's disease (RNAi to remove repeats), amyotrophic lateral sclerosis, epilepsy (galanin, neurotrophic factors) and other neurological disorders, cancer (cytokines including endostatin, angiostatin, TRAIL, FAS-ligand, interferon;RNAi, including RNAi against VEGF or multidrug resistance gene products), diabetes (insulin), muscular dystrophies including Duchenne (dystrophin, mini-dystrophin, insulin-like growth factor I, sarcoglycans [e.g., α, β, γ], RNAi against myostatin, myostatin propeptide, follistatin, activin type II soluble receptor, anti-inflammatory polypeptides, e.g., IkappaB dominant mutant, sarcospan, utrophin, mini-utrophin, RNAi against splice sites in the dystrophin gene that induce exon skipping [see, e.g., WO / 2003 / 095647], antisense to U7 snRNA that induces exon skipping [see, e.g., WO / 2006 / 021724] , and antibodies or antibody fragments against myostatin or myostatin propeptide) and Becker, Gaucher disease (glucocerebrosidase), Hurler disease (α-L-iduronidase), adenosine deaminase deficiency (adenosine deaminase), glycogen storage diseases (e.g., Fabry disease [α-galactosidase] and Pompe disease [lysosomal acid α-glucosidase]) and other metabolic defects, congenital emphysema (α1-antitrypsin), Lesch-Nyhan syndrome (hypoxanthine guanine phosphoribosyltransferase), Niemann-Pick disease (sphingomyelinase), Tay-Sachs disease (lysosomal hexosaminidase A), maple syrup urine disease (branched-chain ketoacid dehydrogenase), retinal degenerative diseases (and other diseases of the eye and retina;for example, PDGF for macular degeneration), diseases of solid organs such as the brain (including: Parkinson's disease [GDNF], astrocytoma [RNAi against endostatin, angiostatin and / or VEGF], glioblastoma [RNAi against endostatin, angiostatin and / or VEGF]), liver, kidney, heart; including congestive heart failure or peripheral arterial disease (PAD) (for example, by delivering: protein phosphatase inhibitor I (I-1), serca2a, di Link finger proteins (regulating the phospholamban gene), Barkct, β2-adrenergic receptor, β2-adrenergic receptor kinase (BARK), phosphoinositide-3 kinase (PI3 kinase), S100A1, parvalbumin, adenylyl cyclase type 6, G protein-coupled receptor kinase type 2 knockdown molecules, e.g., truncated constitutively active bARKct; calsarcin, RNAi against phospholamban;Phospholamban inhibitors or dominant-negative molecules, such as phospholamban S16E, arthritis (insulin-like growth factors), joint disorders (insulin-like growth factor 1 and / or 2), intimal hyperplasia (e.g., by delivering enos, inos), improving heart transplant survival (superoxide dismutase), AIDS (soluble CD4), muscle wasting (insulin-like growth factor I), kidney defects (erythropoietin), anemia (erythropoietin), arthritis (anti-inflammatory factors, e.g., IRAP and TNFα soluble receptor), hepatitis (α-interferon), LDL receptor defects (LDL receptor), hyperammonemia (ornithine transcarbamylase), Krabbe disease (galactocerebrosidase), Batten disease, cerebrospinal ataxias including SCA1, SCA2, and SCA3, phenylketonuria (phenylalanine hydroxylase), autoimmune diseases, etc. The present invention further provides adjuvant therapy for increasing the success of organ transplantation and / or reducing the negative side effects of organ transplantation (e.g., by administering immunosuppressants or inhibitory nucleic acids to block cytokine production). As another example, bone morphogenetic proteins (including BNP2, 7, etc., RANKL and / or VEGF) can be administered with bone allografts, for example, after fracture or surgical removal in cancer patients.

[0179] Gene transfer has substantial potential applications for understanding pathologies and providing therapies. There are several genetic diseases for which vascular genes are known and have been cloned. Generally, these pathologies fall into two classes: deficiency states (usually enzymes), which are generally recessively inherited, and imbalance states, which may involve regulatory or structural proteins and are typically dominantly inherited. For deficiency diseases, gene transfer can be used to deliver normal genes to affected tissues for replacement therapy and to create animal models for the disease using antisense mutations. For imbalance pathologies, gene transfer can be used to create the pathology in a model system, which can then be used in attempts to combat the pathology. Thus, nucleic acid delivery vectors enable the treatment and / or prevention of genetic diseases.

[0180] Nucleic acid delivery vectors can also be used to provide functional nucleic acids to cells in vitro or in vivo.The expression of functional nucleic acids in cells can, for example, reduce the expression of specific target proteins by cells.Therefore, functional nucleic acids can be administered to subjects in need thereof to reduce the expression of specific proteins.

[0181] Nucleic acid delivery vectors find use in diagnostic and screening methods whereby a nucleic acid of interest is transiently or stably expressed in transgenic animal models.

[0182] Nucleic acid delivery vectors can also be used for a variety of non-therapeutic purposes, as will be apparent to those skilled in the art, including, but not limited to, use in protocols for assessing gene targeting, clearance, transcription, translation, etc. Nucleic acid delivery vectors can also be used for the purpose of assessing safety (spread, toxicity, immunogenicity, etc.). Such data are considered, for example, by the U.S. Food and Drug Administration as part of the regulatory approval process prior to evaluation of clinical efficacy.

[0183] In a further aspect, the nucleic acid delivery vector of the present invention can be used to generate an immune response in a subject. According to this embodiment, a nucleic acid delivery vector comprising a nucleic acid sequence encoding an immunogenic polypeptide can be administered to a subject, and an active immune response is mounted by the subject against the immunogenic polypeptide. The immunogenic polypeptide is as described above. In some embodiments, a protective immune response is elicited.

[0184] Alternatively, the nucleic acid delivery vector can be administered to cells ex vivo, and the modified cells can be administered to a subject. The nucleic acid delivery vector containing the nucleic acid is introduced into cells, and the cells are administered to a subject, where the nucleic acid encoding the immunogen can be expressed to induce an immune response in the subject against the immunogen. In certain embodiments, the cells are antigen-presenting cells (e.g., dendritic cells).

[0185] An "active immune response" or "active immunity" is characterized by the participation of host tissues and cells after encountering an immunogen. It involves the differentiation and proliferation of immunocompetent cells within lymphoreticular tissue, resulting in the synthesis of antibodies or the development of cell-mediated responses, or both. Herbert B. Herscowitz, Immunophysiology: Cell Function and Cellular Interactions in Antibody Formation, in IMMUNOLOGY: BASIC PROCESSES 117 (Joseph A. Bellanti ed., 1985). In other words, an active immune response is initiated by the host after exposure to an immunogen by infection or vaccination. Active immunity can be contrasted with passive immunity, which is achieved by the "transfer of preformed substances (antibodies, transfer factors, thymic grafts, interleukin-2) from an actively immunized host to a non-immunized host." Id.

[0186] As used herein, a "protective" immune response or "protective" immunity indicates that the immune response provides some benefit to the subject in terms of preventing or reducing the occurrence of disease. Alternatively, a protective immune response or immunity may be useful in the treatment and / or prevention of disease, particularly cancer or tumors (e.g., by preventing the formation of cancer or tumors, by causing the regression of cancer or tumors, and / or by preventing metastasis, and / or by preventing the growth of metastatic nodules). The protective effect may be complete or partial, so long as the benefits of the treatment outweigh any disadvantages.

[0187] In certain embodiments, the nucleic acid delivery vector or cells containing the nucleic acid can be administered in an immunogenically effective amount as described below.

[0188] Nucleic acid delivery vectors can also be administered for cancer immunotherapy by administering nucleic acid delivery vectors that express one or more cancer cell antigens (or immunologically similar molecules) or any other immunogens that generate an immune response against cancer cells. For example, to treat a patient with cancer and / or prevent cancer from occurring in a subject, an immune response can be generated against a cancer cell antigen in a subject by administering a nucleic acid delivery vector containing a nucleic acid encoding the cancer cell antigen. The nucleic acid delivery vector can be administered to a subject using in vivo or ex vivo methods, as described herein. Alternatively, the cancer antigen can be expressed as part of the nucleic acid delivery vector.

[0189] As another alternative, any other therapeutic nucleic acid (e.g., RNAi) or polypeptide (e.g., cytokine) known in the art can be administered to treat and / or prevent cancer.

[0190] As used herein, the term "cancer" includes tumor-forming cancers. Similarly, the term "cancerous tissue" includes tumors. "Cancer cell antigens" includes tumor antigens.

[0191] The term "cancer" has its art-understood meaning, e.g., the uncontrolled growth of cells that have the potential to spread (i.e., metastasize) to distant sites in the body. Exemplary cancers include, but are not limited to, melanoma, adenocarcinoma, thymoma, lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's lymphoma), sarcoma, lung cancer, liver cancer, colon cancer, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, brain tumor, and any other cancer or malignant condition now known or later identified. In an exemplary embodiment, the present invention provides methods for treating and / or preventing tumor-forming cancer.

[0192] The term "tumor" is also understood in the art to refer, for example, to an abnormal mass of undifferentiated cells within a multicellular organism. Tumors can be malignant or benign. In exemplary embodiments, the methods disclosed herein are used to prevent and treat malignant tumors.

[0193] By the terms "treating cancer," "treatment of cancer," and equivalent terms, it is intended that the severity of cancer is reduced or at least partially eliminated, and / or the progression of the disease is slowed and / or controlled, and / or the disease is stabilized. In certain embodiments, these terms refer to preventing, reducing, or at least partially eliminating metastasis of cancer, and / or preventing, reducing, or at least partially eliminating the growth of metastatic nodules.

[0194] By the terms "cancer prevention" or "preventing cancer" and equivalent terms, it is intended that the method at least partially eliminates or reduces and / or delays the incidence and / or severity of cancer onset. Stated another way, the likelihood or probability of cancer onset in a subject is reduced and / or delayed.

[0195] In certain embodiments, cells can be removed from a subject with cancer and contacted with a nucleic acid delivery vector. The modified cells are then administered to the subject, thereby eliciting an immune response against cancer cell antigens. This method can be advantageously used in immunocompromised subjects who are unable to mount a sufficient immune response in vivo (i.e., unable to produce sufficient amounts of enhanced antibodies).

[0196] It is known in the art that immune responses can be enhanced by immunomodulatory cytokines (e.g., α-interferon, β-interferon, γ-interferon, ω-interferon, τ-interferon, interleukin-1α, interleukin-1β, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, interleukin-12, interleukin-13, interleukin-14, interleukin-18, B-cell growth factor, CD40 ligand, tumor necrosis factor-α, tumor necrosis factor-β, monocyte chemotactic protein-1, granulocyte-macrophage colony-stimulating factor, and lymphotoxin). Thus, immunomodulatory cytokines (preferably CTL-inducing cytokines) can be administered to a subject along with a viral vector.

[0197] Cytokines can be administered by any method known in the art: exogenous cytokines can be administered to a subject, or alternatively, nucleic acids encoding the cytokines can be delivered to a subject using a suitable vector, and the cytokines produced in vivo.

[0198] Subjects, Pharmaceutical Formulations, and Modes of Administration The methods of the present invention find use in both veterinary and medical applications. Suitable subjects include birds, reptiles, amphibians, fish, and mammals. As used herein, the term "mammal" includes, but is not limited to, humans, primates, non-human primates (e.g., monkeys and baboons), cows, sheep, goats, pigs, horses, cats, dogs, rabbits, rodents (e.g., rats, mice, hamsters, etc.), and the like. Human subjects include newborns, infants, adolescents, and adults. Optionally, the subject is "in need" of the methods of the present invention because, for example, the subject has or is believed to be at risk for a disorder, including those described herein, or would benefit from delivery of a polynucleotide, including those described herein. As a further option, the subject may be an experimental animal and / or an animal model of a disease. Preferably, the subject is human.

[0199] In certain embodiments, the heterologous agent and protein M or a functional fragment or derivative thereof are administered to a subject in need thereof as early in the subject's life as possible, for example, as soon as the subject is diagnosed with a disease or disorder. In some embodiments, the method is performed on a newborn subject after the disease or disorder has been identified, for example, by newborn screening. In some embodiments, the method is performed on a subject before the age of 10, for example, before the age of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the method is performed on a juvenile or adult subject after the age of 10. In some embodiments, the method is performed on a fetus in utero after the disease or disorder has been identified, for example, by prenatal screening. In some embodiments, the method is performed on a subject as soon as the subject develops symptoms associated with the disease or disorder. In some embodiments, the method is performed before the subject develops symptoms associated with the disease or disorder, for example, on a subject suspected of having or diagnosed with a disease or disorder but who has not yet begun to show symptoms.

[0200] In certain embodiments, the present invention provides one or more pharmaceutical compositions comprising Protein M or a functional fragment or derivative thereof, alone or in combination with a different agent, in a pharmaceutically acceptable carrier, which may include other medicinal agents, pharmaceutical agents, stabilizers, buffers, carriers, adjuvants, diluents, etc. For injection, the carrier is typically a liquid. For other methods of administration, the carrier may be either a solid or a liquid. For inhalation administration, the carrier is inhalable and may be in solid or liquid particulate form.

[0201] By "pharmaceutically acceptable" is meant a material that is not toxic or otherwise undesirable, ie, the material may be administered to a subject without causing any undesired biological effects.

[0202] One aspect of the present invention is a method for transferring nucleic acids into cells in vitro, e.g., as part of an ex vivo method. A heterologous agent (e.g., a nucleic acid delivery vector, e.g., a viral vector) can be introduced into cells in an appropriate amount, e.g., at a multiplicity of infection according to standard transduction methods appropriate for the particular target cells. The titer of the viral vector to be administered can vary depending on the type and number of target cells and the particular viral vector, and can be determined by one of skill in the art without undue experimentation. In a representative embodiment, the titer is at least about 10 3 infectious units, more preferably at least about 10 5 The infectious units are introduced into the cells.

[0203] The cell(s) into which the nucleic acid delivery vector is introduced can be of any type, including, but not limited to, nervous system cells (including cells of the peripheral and central nervous systems, particularly brain cells, e.g., neurons and oligodendrocytes), lung cells, ocular cells (including retinal cells, retinal pigment epithelium, and corneal cells), vascular cells (e.g., endothelial cells, endothelial cells), epithelial cells (e.g., gastrointestinal and respiratory epithelial cells), muscle cells (e.g., skeletal muscle cells, cardiac muscle cells, smooth muscle cells, and / or diaphragm muscle cells), dendritic cells, pancreatic cells (including pancreatic islet cells), hepatocytes, kidney cells, cardiac muscle cells, bone cells (e.g., bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, germ cells, etc. In exemplary embodiments, the cell can be any progenitor cell. As a further possibility, the cell can be a stem cell (e.g., neural stem cells, liver stem cells). As a further alternative, the cell can be a cancer or tumor cell. Furthermore, the cells can be from any species of origin as described above.

[0204] The nucleic acid delivery vector can be introduced into cells in vitro for the purpose of administering the modified cells to a subject. In certain embodiments, cells are removed from a subject, the nucleic acid delivery vector is introduced into them, and the cells are then administered back to the subject. Methods for removing cells from a subject for ex vivo manipulation and then introducing them back into the subject are known in the art (see, e.g., U.S. Pat. No. 5,399,346). Alternatively, the nucleic acid delivery vector can be introduced into cells from a donor subject, into cultured cells, or into cells from any other suitable source, and the cells are administered to a subject in need thereof (i.e., a "recipient" subject).

[0205] Cells suitable for ex vivo gene delivery are described above. The dosage of cells to be administered to a subject varies depending on the age, condition, and type of the subject, the type of cell, the nucleic acid expressed by the cell, the mode of administration, etc. Typically, at least about 10 2 ~about 10 8 cells or at least about 10 3~about 10 6 The cells are administered per dose in a pharmaceutically acceptable carrier. In certain embodiments, cells transduced with a nucleic acid delivery vector are administered to a subject in a therapeutically or prophylactically effective amount in combination with a pharmaceutical carrier.

[0206] In some embodiments, the nucleic acid delivery vector is introduced into cells, and the cells are administered to a subject to elicit an immunogenic response against the delivered polypeptide (e.g., expressed as a transgene or intracapsidally). Typically, a quantity of cells expressing an immunogenically effective amount of the polypeptide is administered in combination with a pharmaceutically acceptable carrier. An "immunogenically effective amount" is the amount of expressed polypeptide sufficient to elicit an active immune response against the polypeptide in the subject to whom the pharmaceutical preparation is administered. In certain embodiments, the dosage is sufficient to generate a protective immune response (as defined above). The degree of protection conferred need not be complete or permanent, as long as the benefits of administering the immunogenic polypeptide outweigh any disadvantages.

[0207] A further aspect of the present invention is a method for administering a heterologous agent (e.g., a nucleic acid delivery vector) to a subject. The nucleic acid delivery vector can be administered to a human subject or animal in need thereof by any means known in the art. The nucleic acid delivery vector may be delivered in a pharmaceutically acceptable carrier at a therapeutically or prophylactically effective dose.

[0208] The nucleic acid delivery vector can also be administered to induce an immunogenic response (e.g., as a vaccine). Typically, the immunogenic composition of the present invention comprises an immunogenically effective amount of the nucleic acid delivery vector in combination with a pharmaceutically acceptable carrier. The dosage may be sufficient to generate a protective immune response (as defined above). The degree of protection provided need not be complete or permanent, as long as the benefits of administering the immunogenic polypeptide outweigh any disadvantages. The subject and immunogen are as described above.

[0209] The dosage of a nucleic acid delivery vector (e.g., a viral vector) administered to a subject depends on the mode of administration, the disease or condition to be treated and / or prevented, the condition of the individual subject, the particular nucleic acid delivery vector, and the nucleic acid to be delivered, and can be routinely determined. Exemplary doses to achieve a therapeutic effect are at least about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 The titer of transducing units is approximately 10 8 ~10 13 It may also be a transducing unit.

[0210] In certain embodiments, more than one administration (e.g., two, three, four, or more administrations) over various intervals, e.g., daily, weekly, monthly, yearly, etc., may be used to achieve the desired level of gene expression.

[0211] Exemplary modes of administration include oral, rectal, transmucosal, intranasal, inhalation (e.g., via aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intraendothelial, intrauterine (or intraembryonic), parenteral (e.g., intravenous, subcutaneous, intradermal, intracranial, intramuscular (including administration to skeletal muscle, diaphragm, and / or cardiac muscle), intrapleural, intracerebral, and intraarticular), topical (e.g., both to the skin and mucosal surfaces (including respiratory tract surfaces), and transdermal administration), intralymphatic, etc., as well as direct tissue or organ injection (e.g., into the liver, eye, skeletal muscle, cardiac muscle, diaphragm muscle, or brain).

[0212] Administration can be at any site in a subject, including, but not limited to, a site selected from the group consisting of the brain, skeletal muscle, smooth muscle, heart, diaphragm, respiratory epithelium, liver, kidney, spleen, pancreas, skin, and eye.

[0213] Administration may be to the tumor (e.g., within or near the tumor or lymph node). The most suitable route in any given case will depend on the nature and severity of the condition being treated and / or prevented, and on the nature of the particular vector used.

[0214] Administration to skeletal muscles according to the present invention includes, but is not limited to, administration to skeletal muscles in the limbs (e.g., upper arms, forearms, upper thighs, and / or lower legs), back, neck, head (e.g., tongue), chest, abdomen, pelvis / perineum, and / or fingers.Suitable skeletal muscles include, but are not limited to, abductor digiti minimi (hand), abductor digiti minimi (foot), abductor pollicis, abductor digiti minus (5th metatarsus), abductor pollicis brevis, abductor pollicis longus, adductor brevis, adductor hallucis, adductor longus, adductor magnus, adductor pollicis, anconeus, anterior scalene, genu muscles, biceps brachii, biceps femoris, brachialis, brachioradialis, buccinator, coracobrachialis, corrugator supercilii, deltoid, depressor anguli oris, depressor labii inferior, digastric, dorsal interosseous (hand), dorsal interosseous (foot), extensor carpi radialis brevis, extensor carpi radialis longus, extensor carpi ulnaris, extensor digitorum minimi, extensor digitorum brevis, extensor digitorum longus, extensor pollicis brevis, and extensor hallucis longus. Extensor muscles, extensor index muscle, extensor pollicis brevis, extensor pollicis longus, flexor carpi radialis, flexor carpi ulnaris, flexor digitorum brevis (hand), flexor digitorum brevis (foot), flexor digitorum brevis, flexor digitorum longus, flexor digitorum profundus, flexor digitorum superficialis, flexor hallucis brevis, flexor hallucis longus, flexor pollicis brevis, flexor pollicis longus, frontalis muscle, gastrocnemius muscle, geniohyoid muscle, gluteus maximus, gluteus medius, gluteus minimus, gracilis muscle, iliocostalis cervix, iliocostalis lumborum, iliocostalis thoracic, iliacus muscle, inferior gemellus muscle, inferior oblique muscle, inferior rectus muscle, infraspinatus muscle, interspinal muscle, intertransverse muscle, lateral pterygoid muscle, lateral rectus muscle, latissimus dorsi, levator angle oris, infraorbital muscle, levator labii naris superioris, levator palpebrae superioris, levator scapulae, rotator longus, longissimus capitis, longissimus cervix, longissimus thoracis , longus capitis, longus colli, lumbrical muscles (hand), lumbrical muscles (foot), masseter, medial pterygoid, medial rectus, middle scalene, multifidus, mylohyoid, inferior oblique capitis, superior oblique capitis, obturator externus, obturator internus, occipitalis, omohyoid, opponens digiti minimi, opponens pollicis, orbicularis oculi, orbicularis oris, palmar interosseous, palmaris brevis, palmaris longus, pubococcus, pectoralis major, pectoralis minor, peroneus brevis, peroneus longus, peroneus 3, piriformis, plantar interosseous, plantaris, platysma, popliteus, posterior scalene, pronator quadratus, pronator teres, psoas major, quadratus femoris, quadratus plantaris, rectus capitis anterior, rectus capitis lateralis, rectus capitis posterior major, rectus capitis posterior minor, rectus femoris, rhomboid major, rhomboid minor , sartorius, scalenus minimus, semimembranosus, semispinalis capitis, semispinalis cervix, semispinalis thoracicus, semitendinosus, serratus anterior, rotator brevis, soleus, spinalis capitis, spinalis cervix, thoracic spine, splenius capitis, splenius cervix, sternocleidomastoid, sternohyoid, sternothyroid, stylohyoid, subclavius, subscapularis, superior gemellus, superior oblique, superior rectus, supinator, supraspinatus, temporalis, tensor fasciae lata, teres major, teres minor, thoracic cage, thyrohyoid, tibialis anterior, tibialis posterior, trapezius, triceps brachii, vastus intermedius, vastus lateralis, vastus medialis, zygomaticus major and zygomaticus minor, and any other suitable skeletal muscles known in the art.

[0215] Heterologous agents can be delivered to skeletal muscle by intravenous administration, intra-arterial administration, intraperitoneal administration, limb perfusion (which may be isolated limb perfusion of the foot and / or arm; see, e.g., Arruda et al., (2005) Blood 105:3458-3464), and / or direct intramuscular injection. In certain embodiments, heterologous agents are administered to the limbs (arms and / or legs) of a subject (e.g., a subject with muscular dystrophy, such as DMD) by limb perfusion (e.g., by intravenous or intra-articular administration), which may be isolated limb perfusion. In embodiments of the present invention, heterologous agents can be advantageously administered without the use of "hydrodynamic" techniques. Tissue delivery of prior art vectors (e.g., to muscle) is often enhanced by hydrodynamic techniques (e.g., intravenous / intravenous administration in large volumes) that increase pressure within the vasculature and facilitate the ability of the agent to cross the endothelial cell barrier. In certain embodiments, the heterologous agent can be administered in the absence of hydrodynamic techniques, such as large volume infusion and / or elevated intravascular pressure (e.g., above normal systolic pressure, e.g., an increase in intravascular pressure of 5%, 10%, 15%, 20%, 25% or less above normal systolic pressure). Such methods may reduce or avoid side effects associated with hydrodynamic techniques, such as edema, nerve damage, and / or compartment syndrome.

[0216] Administration to the myocardium includes administration to the left atrium, right atrium, left ventricle, right ventricle, and / or septum. The heterologous agent can be delivered to the myocardium by intravenous administration, intra-arterial administration, e.g., intra-aortic administration, direct cardiac injection (e.g., into the left atrium, right atrium, left ventricle, right ventricle), and / or coronary perfusion.

[0217] Administration to the diaphragm muscle can be by any suitable method, including intravenous administration, intraarterial administration, and / or intraperitoneal administration.

[0218] Administration to smooth muscle can be by any suitable method, including intravenous, intraarterial, and / or intraperitoneal administration. In one embodiment, administration can be to endothelial cells located within, near, and / or on smooth muscle.

[0219] Delivery to the target tissue can also be achieved by delivering a depot containing the heterologous agent. In an exemplary embodiment, a depot containing the heterologous agent is implanted into skeletal, smooth, cardiac, and / or diaphragm muscle tissue, or the tissue can be contacted with a film or other matrix containing the heterologous agent. Such implantable matrices or substrates are described in U.S. Patent No. 7,201,898.

[0220] In certain embodiments, the heterologous agent is administered to skeletal muscle, diaphragm muscle, and / or cardiac muscle (eg, to treat and / or prevent muscular dystrophy or heart disease (eg, PAD or congestive heart failure)).

[0221] In an exemplary embodiment, the present invention is used to treat and / or prevent disorders of skeletal muscle, cardiac muscle and / or diaphragm muscle.

[0222] In an exemplary embodiment, the present invention provides a method for treating and / or preventing muscular dystrophy in a subject in need thereof, the method comprising administering to a mammalian subject a therapeutically or prophylactically effective amount of a heterologous agent, wherein the heterologous agent comprises dystrophin, mini-dystrophin, micro-dystrophin, myostatin propeptide, follistatin, activin type II soluble receptor, IGF-1, an anti-inflammatory polypeptide such as an IkappaB dominant mutant, sarcospan, utrophin, micro-dystrophin, laminin-α2, α-sarcoglycan, β-sarcoglycan, γ-sarcoglycan, δ-sarcoglycan, IGF-1, an antibody or antibody fragment against myostatin or myostatin propeptide, and / or a nucleic acid encoding an RNAi against myostatin. In certain embodiments, the heterologous agent can be administered to skeletal muscle, diaphragm muscle, and / or cardiac muscle, as described elsewhere herein.

[0223] Alternatively, the present invention can be practiced to deliver nucleic acids to skeletal muscle, cardiac muscle, or diaphragm muscle to be used as a platform for the production of polypeptides (e.g., enzymes) or functional nucleic acids (e.g., functional RNAs, e.g., RNAi, microRNAs, antisense RNAs) that normally circulate in the blood or for systemic delivery to other tissues to treat and / or prevent disorders (e.g., metabolic disorders, e.g., diabetes (e.g., insulin), hemophilia (e.g., factor IX or factor VIII), mucopolysaccharidosis (e.g., Sly syndrome, Hurler syndrome, Scheie syndrome, Hurler-Scheie syndrome, , Hunter syndrome, Sanfilippo syndrome A, B, C, D, Morquio syndrome, Maroteaux-Lamy syndrome, etc.) or lysosomal storage diseases (e.g., Gaucher disease [glucocerebrosidase], Pompe disease [lysosomal acid α-glucosidase], or Fabry disease [α-galactosidase A]) or glycogen storage diseases (e.g., Pompe disease [lysosomal acid α-glucosidase]). Other suitable proteins for treating and / or preventing metabolic disorders are described above. The use of muscle as a platform for expressing a nucleic acid of interest is described in U.S. Patent Publication No. 2002 / 0192189.

[0224] Thus, in one aspect, the present invention further encompasses a method for treating and / or preventing a metabolic disorder in a subject in need thereof, the method comprising administering a therapeutically or prophylactically effective amount of a heterologous agent to the subject (e.g., to the skeletal muscle of the subject), wherein the heterologous agent comprises a nucleic acid encoding a polypeptide, and wherein the metabolic disorder is the result of a deficiency and / or defect in the polypeptide. Exemplary metabolic disorders and nucleic acids encoding the polypeptides are described herein. The polypeptide may be secreted (e.g., a polypeptide that is a secreted polypeptide in its natural state, or a polypeptide that has been modified to be secreted, e.g., by operative association with a secretory signal sequence known in the art). Without being limited to any particular theory of the invention, according to this embodiment, administration to skeletal muscle can result in the secretion of the polypeptide into the systemic circulation and delivery to the target tissue(s). Methods for delivering a heterologous agent to skeletal muscle are described in more detail herein.

[0225] The present invention can also be practiced to produce antisense RNA, RNAi or other functional RNA (eg, ribozymes) for systemic delivery.

[0226] The present invention also provides a method of treating and / or preventing congenital heart defect or PAD in a subject in need thereof, the method comprising administering to the mammalian subject a therapeutically or prophylactically effective amount of a heterologous agent of the present invention, wherein the heterologous agent is, for example, a heterologous agent that inhibits sarcoplasmic endoreticulum Ca2+ receptors. 2+-ATPase (SERCA2a), angiogenesis factors, phosphatase inhibitor I (I-1), RNAi against phospholamban; phospholamban inhibitory or dominant-negative molecules, such as phospholamban S16E, zinc finger proteins that regulate the phospholamban gene, β2-adrenergic receptor, β2-adrenergic receptor kinase (BARK), PI3 kinase, calsarcan, β-adrenergic receptor kinase inhibitor (βAR) Kct), inhibitor of protein phosphatase 1, S100A1, parvalbumin, adenylyl cyclase type 6, molecules that result in G protein-coupled receptor kinase type 2 knockdown, such as nucleic acids encoding truncated constitutively active bARKct, Pim-1, PGC-1α, SOD-1, SOD-2, EC-SOD, kallikrein, HIF, thymosin-β4, mir-1, mir-133, mir-206, and / or mir-208.

[0227] Injectables can be prepared in conventional forms, such as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid before injection, or emulsions. Alternatively, the heterologous agent can be administered in a local rather than systemic manner, for example, in a depot or sustained-release formulation. Furthermore, the heterologous agent can be delivered by being attached to a surgically implantable matrix (for example, as described in U.S. Patent Publication No. 2004-0013645).

[0228] The heterologous agents disclosed herein can be administered to a subject's lungs by any suitable means, including administration of an aerosol suspension of respirable particles comprised of the heterologous agent, which the subject inhales. The respirable particles can be liquid or solid. Aerosols of liquid particles containing the heterologous agent can be produced by any suitable means, as known to those skilled in the art, for example, using a pressure-driven aerosol nebulizer or an ultrasonic nebulizer. See, e.g., U.S. Pat. No. 4,501,729. Aerosols of solid particles containing the heterologous agent can similarly be produced using any solid particulate drug aerosol generator, according to techniques known in the pharmaceutical arts.

[0229] Heterologous agents can be administered to tissues of the CNS (eg, brain, eye), which may advantageously result in a wider distribution of the heterologous agent than would be observed in the absence of the present invention.

[0230] In certain embodiments, heterologous agents may be administered to treat diseases of the CNS, including genetic disorders, neurodegenerative disorders, psychiatric disorders, and tumors. Exemplary diseases of the CNS include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan disease, Leigh's disease, Refsum's disease, Tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, trauma due to spinal cord or head injury, Tay-Sachs disease, Lesch-Nyan disease, epilepsy, stroke, and psychiatric disorders, including mood disorders (e.g., depression, bipolar disorder, persistent affective disorder). , secondary mood disorders), schizophrenia, drug addiction (e.g., alcoholism and other substance addictions), neuroses (e.g., anxiety, obsessional disorders, somatoform disorders, dissociative disorders, grief, postpartum depression), psychoses (e.g., hallucinations and delusions), dementia, paranoia, attention deficit disorder, psychosexual disorders, sleep disorders, pain disorders, eating or weight disorders (e.g., obesity, cachexia, anorexia nervosa, and bulimia), and cancers and tumors of the CNS (e.g., pituitary tumors).

[0231] Disorders of the CNS include ocular disorders involving the retina, posterior tract, and optic nerve (e.g., retinitis pigmentosa, diabetic retinopathy and other retinal degenerative diseases, uveitis, age-related macular degeneration, glaucoma).

[0232] Most, if not all, ocular diseases and disorders are associated with one or more of three types of indications: (1) angiogenesis, (2) inflammation, and (3) degeneration. The heterologous agents of the present invention can be used to deliver anti-angiogenic factors; anti-inflammatory factors; factors that slow cell degeneration, promote cell sparing, or promote cell proliferation, and combinations of the foregoing.

[0233] Diabetic retinopathy, for example, is characterized by neovascularization. Diabetic retinopathy can be treated by delivering one or more anti-angiogenic factors intraocularly (e.g., intravitreally) or periocularly (e.g., sub-Tenon's region). One or more neurotrophic factors may also be co-delivered intraocularly (e.g., intravitreally) or periocularly.

[0234] Uveitis involves inflammation. One or more anti-inflammatory factors can be administered by intraocular (e.g., intravitreal or anterior chamber) administration of a delivery vector of the invention.

[0235] Retinitis pigmentosa, by comparison, is characterized by retinal degeneration. In an exemplary embodiment, retinitis pigmentosa can be treated by intraocular (e.g., intravitreal) administration of a heterologous agent encoding one or more neurotrophic factors.

[0236] Age-related macular degeneration involves both neovascularization and retinal degeneration. This disorder can be treated by administering heterologous agents encoding one or more neurotrophic factors intraocularly (e.g., in the vitreous) and / or one or more anti-angiogenic factors intraocularly or periocularly (e.g., in the sub-Tenon's area).

[0237] Glaucoma is characterized by increased intraocular pressure and loss of retinal ganglion cells. Treatment for glaucoma involves administering one or more neuroprotective agents to protect cells from excitotoxic damage using heterologous drugs. Such drugs include N-methyl-D-aspartate (NMDA) antagonists, cytokines, and neurotrophic factors, which are delivered intraocularly (possibly intravitreally).

[0238] In other embodiments, the present invention can be used to treat seizures, e.g., to reduce the onset, incidence, or severity of seizures. The efficacy of therapeutic treatment for seizures can be assessed by behavioral measures (e.g., eye or mouth tremors, tics) and / or electrographic measures (most seizures have signature electrographic abnormalities). Thus, the present invention can also be used to treat epilepsy characterized by multiple seizures over time.

[0239] In one exemplary embodiment, somatostatin (or an active fragment thereof) is administered to the brain using a heterologous agent of the present invention to treat pituitary tumors. According to this embodiment, a heterologous agent encoding somatostatin (or an active fragment thereof) is administered to the pituitary gland by microinjection. Similarly, such treatment can be used to treat acromegaly (abnormal growth hormone secretion from the pituitary gland). The nucleic acid (e.g., GenBank Accession No. J00306) and amino acid (e.g., GenBank Accession No. P01166; including the processed active peptides somatostatin-28 and somatostatin-14) sequences of somatostatin are known in the art.

[0240] In certain embodiments, the heterologous agent can include a secretion signal as described in US Pat. No. 7,071,172.

[0241] In a representative embodiment of the present invention, a heterologous agent is administered to the CNS (e.g., the brain or eye). The heterologous agent may be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (striatum, cerebrum, including: occipital, temporal, parietal, and frontal lobes, cortex, basal ganglia, hippocampus, and amygdala), limbic system, neocortex, striatum, cerebrum, and inferior colliculus. The heterologous agent may also be administered to different regions of the eye, such as the retina, cornea, and / or optic nerve.

[0242] Heterologous agents can be delivered to the cerebrospinal fluid (e.g., by lumbar puncture) for more dispersed administration of the heterologous agent. Heterologous agents can also be administered intravascularly to the CNS in situations where the blood-brain barrier is disrupted (e.g., brain tumor or cerebral infarction).

[0243] Heterologous agents can be administered to the desired region(s) of the CNS by any route known in the art, including, but not limited to, intrathecal, intraocular, intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intraaural, intraocular (e.g., intravitreal, subretinal, anterior chamber) and periocular (e.g., sub-Tenon's area) delivery, and intramuscular delivery by retrograde delivery to motor neurons.

[0244] In certain embodiments, the heterologous agent is administered in a liquid formulation to the desired region or compartment within the CNS by direct injection (e.g., stereotactic injection). In other embodiments, the heterologous agent can be provided by topical application to the desired region or by intranasal administration of an aerosol formulation. Administration to the eye can be by topical application of liquid drops. As a further alternative, the heterologous agent can be administered as a solid sustained-release formulation (see, e.g., U.S. Patent No. 7,201,898).

[0245] In further embodiments, heterologous agents can be used for retrograde transport to treat and / or prevent diseases and disorders involving motor neurons (e.g., amyotrophic lateral sclerosis (ALS); spinal muscular atrophy (SMA), etc.). For example, heterologous agents can be delivered to muscle tissue, from which they can be translocated to neurons.

[0246] Protein M or a functional fragment or derivative thereof may be administered by any route or schedule described above for the heterologous agent. Protein M or a functional fragment or derivative thereof may be administered by a different route or schedule than the heterologous agent.

[0247] Having described the invention, the same is described in more detail in the following examples, which are included herein for illustrative purposes only and are not intended to be limitations on the invention. [Example]

[0248] Example 1: Methods AAV virus production AAV vectors were produced using standard three-plasmid transfection techniques in HEK293 cells. Briefly, the AAV transgene plasmid pTR-CBA-Luc was cotransfected with the AAV Rep / Cap helper plasmid (pXR2 or pXR8) and the adenovirus helper plasmid pXX6-80. After 72 hours, cell cultures were harvested and lysed by freeze-thawing and sonication. The clarified cell lysates were DNAse-treated, ultracentrifuged in a 15% / 25% / 40% / 60% iodixanol step gradient, and purified on an anion-exchange Q-column. The purified AAV vectors were titrated by qPCR using primers designed to amplify segments of the packaged AAV transgene.

[0249] Protein production Plasmid pET-28b(+), encoding protein M (a truncated M. genitalium protein MG281 (amino acids 74–479) lacking the transmembrane domain) and carrying an N-terminal His-Tag and thrombin cleavage site, was kindly provided by Rajesh. The plasmid was propagated in electrocompetent DH10B cells and purified using the PureLink Maxi-Prep kit from Invitrogen. The pET-28b(+) plasmid was transiently transfected into BL21 / DE3 cells using an overnight starter culture. Autoinduction medium (Magic medium from Invitrogen) was inoculated with the starter culture and grown at 18°C ​​for 3 days in a 1–4 L culture volume. The culture was then pelleted by centrifugation and frozen at −80°C.

[0250] Protein purification Frozen bacterial cell culture pellets were thawed, lysed by sonication, DNAse-treated, and clarified by centrifugation. The clarified bacterial lysate was dialyzed into nickel binding buffer (20 mM imidazole, 50 mM sodium phosphate (pH 7.4), 500 mM NaCl, 0.02% sodium azide) and passed through a nickel His-trap FF column using FPLC. Protein M bound to the nickel column was then eluted with the same buffer base but containing 500 mM imidazole. Protein M was then passed through an S-100 size-exclusion column, dialyzed into phosphate-buffered saline containing 2% glycerol, and quantified spectrophotometrically. The identity of Protein M was confirmed using SDS-PAGE protein gel electrophoresis for protein separation, followed by Coomassie blue protein staining, which correctly identified the 48 kDa Protein M band.

[0251] Western blot After protein separation on an 8% SDS-PAGE gel, proteins were transferred onto a PVDF membrane. Immunoblotting was performed using a primary anti-His antibody at a 1:1000 dilution (10 μg / ml) in 5% nonfat milk. A secondary goat anti-human IgG antibody was conjugated to horseradish peroxidase (1:10,000 dilution).

[0252] cell culture HEK-293 and Huh7 cells were used for all in vitro AAV neutralization experiments and transduction enhancement, respectively. Cells were maintained at 37°C in 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin-streptomycin.

[0253] Human IVIG and immunized mouse serum 10% human IVIG (Gamunex) was purchased from Grifols Therapeutics Inc. (Research Triangle Park, NC, USA). Serum from 12 different mice (50% male, 50% female) was collected at 3 × 10 10The viral genome, AAV8-FVIII, was administered IP, followed by a boost with the same vector 2 weeks later, and a second boost 6 weeks after the first dose, after which the mice were harvested and pooled. Human IVIG and mouse serum were aliquoted and stored at -80°C for future use.

[0254] In vitro AAV neutralization assay NAb analysis was performed as previously described with slight modifications. Cells were pelleted by centrifugation, resuspended in serum-free X-Vivo 10 medium, and then plated in either 48- or 96-well plate formats. Human IVIG or serum was serially diluted 2-fold or 10-fold. AAV-Luc was incubated with human IVIG or mouse serum for 1 hour at 4°C, followed by the addition of AAV and an additional 1 hour at 4°C. The AAV + serum incubation was then mixed with cells suspended in serum-free medium at the time of plating. In neutralization experiments using Protein M, three different incubation scenarios were tested at each step for 1 hour at 4°C: neutralizing serum was first incubated with Protein M followed by AAV; AAV was first incubated with neutralizing serum followed by Protein M; and AAV was first incubated with Protein M followed by neutralizing serum. Cells were seeded in 48-well plates in 200 μl medium or 96-well plates in 100 μl medium. After transduction, cells were cultured at 37°C for 24–48 h to allow expression of the AAV-luciferase transgene. To measure Luc activity, cells were lysed with passive lysis buffer (Promega, Madison, WI, USA), and the luciferase signal was measured using a Wallac 1420 Victor 2 automated plate reader. NAb titers were defined as the highest dilution at which luciferase activity was 50% lower than that of serum-free controls.

[0255] In vivo AAV neutralization and passive transfer of NAb seraC57BL / 6 mice were injected with different amounts of serum containing AAV NAbs into the retro-orbital vein, followed by AAV injection 20 minutes later. For mice receiving Protein M treatment prior to AAV administration, Protein M (6.3 mg for the 2:1 ratio, 3.15 mg for the 1:1 ratio, or 1.58 mg for the 0.5:1 ratio) was delivered via the retro-orbital vein 5–15 minutes after serum injection. AAV was administered at a dose of 2 × 10 12 Five minutes later, the AAV-Luc vector was administered by systemic injection of 100 particles / kg. Imaging was performed 1 day after AAV administration and at 1 week and 9 days after injection.

[0256] Example 2: Protein M ablates the inhibitory activity of IVIG against AAV transduction To test Protein M's ability to block antibodies, an in vitro AAV neutralization assay using human intravenous immunoglobulin gamma (IVIG) was established. AAV neutralization assays were performed using serial dilutions of IVIG to determine the amount of IVIG required to neutralize a given amount of AAV2. Luciferase activity produced by cell transduction with an AAV-luciferase vector served as a functional readout of gene expression. Neutralization was determined as a percentage of luciferase activity normalized to a no-IVIG control. 12.5 μg of IVIG was found to neutralize 75% (+ / - 5%) of AAV2 (Figure 1), and this amount of IVIG was selected to advance the experimental design to test Protein M, which blocks IVIG, for AAV neutralization. Next, a dose-dilution series of Protein M (SEQ ID NO: 2), recovered by thrombin cleavage to remove the His tag, was performed to examine its ability to block 12.5 μg of IVIG (Figure 2). Protein M was incubated with IVIG for 1 hour, followed by AAV2 for 1 hour at 4°C before transduction of cell cultures. A molar ratio of two molecules of Protein M to one molecule of IgG was found to be sufficient to prevent neutralization to a level comparable to the no-IVIG control. Furthermore, higher molar ratios of Protein M to IVIG were found to enhance the luciferase signal to a level higher than the no-IVIG control (Figure 2). Eight molecules of Protein M to one molecule of IgG increased luciferase expression twofold. To see whether higher molar ratios would further increase the enhancement, 8:1 and 20:1 ratios of Protein M to IVIG were tested under the same experimental conditions. Increasing the Protein M dose by 2.5-fold (from an 8:1 to a 20:1 ratio) provided little further enhancement, as the corresponding signal increased only 0.25-fold relative to the 8:1 ratio (Figure 3). Furthermore, the immunoglobulin-blocking activity of Protein M was found to be independent of cleavage of the N-terminal His-tag; therefore, Protein M without cleaved His-tag was used in subsequent experiments.

[0257] Example 3: Interaction of Protein M with AAV Vector Virions Enhances AAV Transduction Previous studies by the present inventors have shown that the interaction of AAV virions with serum proteins can enhance AAV transduction. To further characterize the enhancing function of Protein M on AAV transduction, a dose-response assay was performed in which two-fold serial dilutions of Protein M were incubated with AAV for 1 hour without IVIG prior to transduction of cell cultures. Figure 4 shows that Protein M at an 8:1 ratio (33 μg Protein M) from previous experiments can dose-dependently enhance AAV transduction in the absence of IVIG, and that the enhancement was greater than 2 × 10 8We found that protein M was lost at dilutions below 2 μg relative to viral particles. The equivalent molar ratio of protein M molecules to AAV particles at which enhancement was lost was less than 40,000:1. Next, we demonstrated that protein M enhancement of transduction depends on the incubation of AAV with protein M. Figure 5 shows cell culture transduction assays in which protein M was added to AAV 1 h before addition to cells (preincubation, -1 h time point), at the time of transduction without preincubation (peri-transduction, 0 h time point), or 18 h after AAV was added to cells (post-transduction, 18 h time point). Similar to Figure 4, a dose-dependent enhancement was observed in the preincubation group but not in the other two groups in which protein M and AAV2 were not incubated together prior to the assay. Furthermore, these results indicate that the mechanism of protein M enhancement involves interactions with the vector capsid and not a biological effect of protein M on the cells, since adding protein M to cells at the time of transduction had no effect on luciferase signal. Finally, we demonstrated that protein M was unable to block neutralization when IVIG was first preincubated with AAV2 for 1 hour followed by a 1-hour incubation with protein M, and that enhancement of AAV2 luciferase signal by protein M only occurred when AAV2 was not fully neutralized by IVIG. For this assay, we used 12.5 μg of IVIG (neutralizing 75–80% of AAV2), 50 μg of IVIG (neutralizing 99% of AAV2, Figure 1), or 200 μg of IVIG (neutralizing 100% of AAV2). In Figure 6, we observed that when 99-100% of AAV2 was neutralized by IVIG (50 μg and 200 μg), Protein M was unable to block neutralization and confer enhanced transduction. Combined with the data in Figures 2 and 3, this result suggests that excess amounts of Protein M after binding to immunoglobulins are able to interact with AAV virions, resulting in enhanced transduction.

[0258] Example 4: Pre-incubation of AAV vector virions with protein M protects IVIG from AAV neutralization Next, instead of first interacting Protein M with IVIG, we decided to test the effect of preincubating Protein M with AAV and then incubating with IVIG. We tested the ability of Protein M to block neutralization when Protein M was incubated with AAV2 for 1 hour, followed by the addition of IVIG and an additional hour at 4°C before cell transduction. Figure 7 shows that when the Protein M concentration was kept unchanged (8.25 μg) and the IVIG dose was serially diluted from 50 μg to 3.12 μg (1:2 to 8:1 molar ratio of Protein M to IVIG), vector protection from neutralization was achieved at a ratio of 2:1 or greater. This is similar to previous results when Protein M was incubated with IVIG before adding AAV. In an attempt to more closely simulate the in vivo situation, where other types of proteins besides IgG are present in serum, similar in vitro neutralization studies were performed in the presence of 10% fetal bovine serum (FBS). According to the manufacturer's IgG quantification for bovine serum, the medium volume was estimated to contain approximately 350 μg of bovine IgG per well. Then, 25 μg of IVIG or serum-free medium was spiked into half of the wells to neutralize AAV or serve as a control without neutralizing activity. In Figure 8, Protein M was incubated with AAV for 1 hour prior to transduction, and total IgG (human and bovine) was added at different molar ratios of 4:1, 2:1, and 1:1 (Protein M to IgG). Protection from neutralization was observed even at the 1:1 ratio. These results demonstrate that Protein M is capable of binding and blocking immunoglobulins, even in the presence of other serum proteins.

[0259] Example 5: Protein M efficiently blocks the neutralizing activity of murine serum against AAV in vitro To translate the IVIG findings into a practical situation, in vitro neutralization experiments were first performed with an AAV8-luciferase vector using serum from mice immunized with an AAV8 capsid vector. When both serum from immunized mice and Protein M were serially diluted to maintain a 2:1 molar ratio, results compared with a serum-only control showed that Protein M prevented escape from neutralization, with a neutralizing serum titer of 1:2,564, an approximate 100-fold dilution (50% neutralization required 0.0039 μl of serum compared to 0.2744 μl of serum in the presence of Protein M; Figure 9).

[0260] Example 6: Protein M effectively blocks the neutralizing activity of murine serum against AAV in vivo Next, in vivo experiments were performed in which different volumes of neutralizing serum were passively transferred into naive mice via retro-orbital injection. After serum perfusion of the mice for 5–15 min, Protein M was administered systemically to the mice, also via retro-orbital injection, followed 5 min later by the administration of AAV8-Luc (2 × 10 10 The mice were administered 100 μl of AAV8-Luc (viral genome). Figure 10 shows that an approximate molar ratio of 2:1 (6.3 mg) of Protein M to total immunoglobulin in mice can prevent neutralization of AAV8 by 1 μl of AAV8-NAb serum (titer 1:2,564). This contrasts with in vivo neutralization assays of serially diluted anti-AAV8 serum, in which greater than 50% of AAV8-Luc was neutralized in serum volumes ranging from 1 μl to 0.001 μl, representing over a 1,000-fold difference in AAV NAb escape (Figure 11).

[0261] Example 7: Stability of Protein M / immunoglobulin complexes To test how stable the immunoglobulin-Protein M complexes were, an assay was first performed in vitro. Protein M was preincubated with mouse serum at a 2:1 molecular ratio for different durations, ranging from 1 hour to 72 hours, at 37°C. Then, AAV8 vectors were added for an additional hour at 4°C for neutralization analysis. As shown in Figure 12, preformed complexes between anti-AAV8 immunoglobulins in mouse serum and Protein M were stable for more than 72 hours when incubated at 37°C before being added to cell cultures, compared to controls containing neutralizing serum but no Protein M, or controls containing PBS or medium alone. This result suggests that the complexes formed between Protein M and immunoglobulins are highly stable in vitro.

[0262] Next, the in vivo stability of Protein M was tested after passive transfer of 0.3 μl of AAV8-neutralizing serum. When Protein M was administered and 3 hours was allowed to pass instead of 5 minutes, the ability of Protein M to block antibodies was reduced by 3.5 × 10 5 From 1×10 5 photons / second / cm 2 The spontaneous rate of NAb-blocking activity was found to be reduced by approximately 70% (FIG. 13). This result indicates that the NAb-blocking effect of Protein M is short-lived in vivo.

[0263] Gene therapy using AAV vectors has been successful in clinical trials. However, the high incidence of AAV-neutralizing antibodies in the human population prevents more patients from benefiting from this effective gene delivery. In this study, we found that mycoplasma-derived protein M can interact with NAb and enhance AAV transduction. The minimum dose of protein M required to block NAb activity was 2-fold more than immunoglobulin molecules. Direct interaction of protein M with AAV virions also enhanced AAV transduction. Neutralizing antibody assays showed that NAb activity was reduced approximately 100-fold when serum from AAV-immunized mice was incubated with protein M in vitro, and over 1000-fold protection was observed in mice adoptively transferred with NAb-positive serum when protein M was administered. Although complexes of immunoglobulins and Protein M were stable over time in vitro, Protein M gradually lost its protective function from immunoglobulin neutralization in vivo.

[0264] To overcome AAV NAb resistance, several strategies have been utilized in laboratories. One approach is to mask the AAV surface to block NAb recognition using coating polymers or exosomes. While this approach is promising, it may alter the AAV transduction profile. A second approach is to generate a library of AAV capsid mutations using error-prone PCR or DNA shuffling and select NAb escape mutants in vitro and in vivo in the presence of NAb. While this approach has produced novel capsids, it has the potential limitation of producing capsids with unknown transduction efficiencies in humans because these mutants are isolated from and tested in animal tissues. Data from animal testing is not always transferable to humans, and no authentic system is available to predict AAV transduction in human tissues. A third approach is to use alternative AAV serotypes with low or no NAb cross-reactivity. Although this popular strategy is logical and successful in animal models, concerns remain about the presence of cross-reactivity in most humans, which cannot be predicted in animals. A final experimental approach is to rationally modify the NAb-binding domain on the AAV capsid surface to eliminate the NAb-binding site. This strategy requires information about monoclonal antibody epitopes and the structure of the AAV virion, and is inherently limited by the fact that NAb derived from human serum is polyclonal, making it impossible to obtain human-derived mAbs that represent all produced NAb. Several clinically relevant approaches are also being investigated; one example is plasma exchange prior to vector delivery. However, due to the relative inefficiency of each apheresis and the fact that even low-titer NAb (<1:5) can inhibit AAV transduction, this strategy is only appropriate for patients with lower starting AAV NAb titers and requires multiple apheresis sessions.Similarly, the use of anti-CD20 antibodies (rituximab) can achieve B cell depletion, but this process takes a long time (approximately 6–9 months) and is only effective in reducing AAV NAb in a small number of subjects. A final clinical approach is to use excess empty AAV capsids as decoys for NAb. The addition of empty particles increases the AAV capsid load, potentially increasing the elimination of AAV-transduced cells mediated by capsid-specific cytotoxic immune responses, possibly competing with intact AAV particles for effective transduction. Furthermore, empty capsids can induce greater liver inflammation than intact AAV vectors. Compared to the low efficiency of NAb protection or the complexity of the above approaches, this study demonstrated a strategy with greater potential for blocking neutralizing antibody activity using immunoglobulin-binding protein M. NAb evasion was achieved with Protein M for 100-fold in vitro and over 1000-fold in vivo.

[0265] Protein M functions as a universal antibody-binding protein that blocks mammalian IgG, IgM, and IgA antibody classes by universally binding to conserved regions on antibody light and heavy chains, resulting in structural interference with antigen recognition or CDR regions. Protein M binds to antibodies, preventing antigen-antibody binding but not disrupting pre-formed antigen-antibody complexes. The interaction between antibodies and Protein M has been verified by Western blot, ELISA, X-ray crystallography, electron microscopy, and biolayer interferometry. Protein M can be used independently of vectors, allowing it to be incorporated into treatment regimens, including previously FDA-approved gene therapies. This offers an advantage over capsid-based immune evasion, since each individual capsid must undergo its own clinical trials for each disease target. Based on the properties of Protein M, this protein can be applied not only to any viral vector-mediated gene delivery but also to transient protein therapies, such as CRISPR / Cas9, to circumvent humoral immune response-mediated clearance. Protein M also has the potential to treat autoimmune disorders caused by autoantibodies.

[0266] This study demonstrated that at least two or more molecules of Protein M are required to block one immunoglobulin molecule, and that a large amount of Protein M may be required to interact with all immunoglobulins in the blood for NAb evasion after systemic administration. High doses of recombinant proteins, such as human serum albumin and immunoglobulins, have been used in clinical trials, but concerns about potential complications of high doses of Protein M need to be addressed in large animal studies before clinical trials. This may not be a major issue if Protein M is used locally for AAV administration.

[0267] In vitro studies have shown that Protein M / immunoglobulin complexes are relatively stable over long periods of time, but in vivo experiments have shown a gradual loss of Protein M function when AAV vectors are administered long after Protein M application. Understanding the dynamics and kinetics of Protein M / immunoglobulin complexes in the blood is important; this information provides valuable information regarding the window for AAV infusion after Protein M administration.

[0268] Another concern is the immunogenicity of Protein M with repeated administration. Because Protein M is a foreign protein, it induces a humoral immune response, resulting in the production of antibodies. Protein M exerts its protective function by binding to all immunoglobulins, and the amount of specific Ig against Protein M is a very small fraction of all Ig. Therefore, when high doses of Protein M are used to block AAV NAb, the amount of specific Ig neutralizes only a very small amount of Protein M, and therefore, the administered Protein M may not affect its ability to protect AAV from AAV NAb. Protein M can bind to the B cell surface and has the potential to stimulate B cell proliferation. Previous studies have shown that intact Protein M can induce B cell proliferation, but truncated Protein M loses this function. Long-term in vivo follow-up after Protein M administration is warranted.

[0269] In conclusion, these results demonstrate that Protein M prevents immunoglobulins from neutralizing AAV when present at a molecular ratio of two or more Protein M molecules to one IgG molecule. Protection of AAV vectors relies on Protein M interacting with immunoglobulins prior to immunoglobulin neutralization of AAV. Protein M can protect AAV from neutralization in vivo across a 1,000-fold range of NAb titers. This study provides important insights into the use of Protein M to protect AAV vector virions from NAb activity in future AAV clinical trials in patients with NAbs or for rechallenge.

[0270] Example 8: Modified Protein M variants with enhanced properties The above example describes the use of mycoplasma protein M as an antibody-blocking protein to circumvent neutralizing antibodies against gene therapy vectors. Although naturally occurring protein M homologs from different mycoplasma species bind with nanomolar affinity to most mammalian antibody classes (Grover et al., Science 343:656 (2014)), many characteristics of the naturally occurring protein make it unsuitable for use as a therapeutic. Native protein M is not soluble but is membrane-bound by an N-terminal transmembrane domain that anchors it within the bacterial cell membrane. Furthermore, the native protein possesses a disordered C-terminus that could behave unexpectedly as a drug-like molecule. A truncated form of native Protein M (Protein M TD by Grover et al., Science 343:656 (2014)), lacking the N-terminal transmembrane domain and disordered C-terminal segment, was tested as a therapeutic antibody blocking molecule, but the key finding was that the protein lacked structural stability when incubated at body temperature (37°C).

[0271] Exposure of truncated protein M (SEQ ID NO: 3) to 37°C in a simple in vitro buffer suspension resulted in visible precipitation and aggregation within a short period (15 min to 1 h). Analysis using circular dichroism revealed that heating the protein to 37°C was associated with a clear protein unfolding event within 1 h, with immediate melting of the protein occurring at 41.2°C, whereas continued incubation at 20°C for 2 h resulted in no unfolding event (Figure 14). Protein unfolding also correlated with a measured reduction in the soluble fraction in solution, as assessed by Western blot. To overcome the therapeutic limitations of using unstable proteins, a rational design approach was used to generate mutant analogs of truncated protein M, including mutant homologs from M. genitalium (MG281) and M. pneumoniae (MPN400). This approach used the crystal structure and amino acid sequence of protein M (PDB IDs: 4NZR and 4NZT) and engineering software called Rosetta as input for protein modeling. Free energy calculations and 3D models were used to predict which amino acid sequence changes would stabilize the tertiary structure, resulting in a list of over 850 amino acid substitutions. Rationally designed mutation libraries were used to generate mutant proteins through DNA synthesis of individual and combined mutants. Table 4 lists the 885 computationally identified point mutations that had better thermostability scores than the wild-type protein. Table 5 lists the 165 computationally identified point mutations that had substantially better thermostability scores than the wild-type protein (better than a -3.0 delta score).

[0272] Differential scanning fluoroscopy was used to examine different protein M mutants and calculate protein melting temperatures (Figures 15 and 16). The mutants were then subjected to 37°C temperature exposure for different periods to measure solubility (Figures 17A-17C) and their ability to prevent AAV neutralization by pooled human intravenous IgG (IVIG) in an in vitro neutralization assay (Figures 18A-18C). In addition to testing analogs of protein M derived from M. genitalium, analogs derived from M. pneumoniae were also produced. Several mutants were then tested by biolayer interferometry (BLI) to determine the affinity of the mutant proteins for mouse IgG, demonstrating that the mutants can alter the affinity of the mutants for immunoglobulin substrates (Figures 21 and 22). The mutants also showed increased stability, as evidenced by their stability over a wider pH range than wild-type protein M (Figure 26). MG29, one of the lead analogs with increased stability at 37°C and maintaining nanomolar affinity for IgG, was used to test blocking of AAV neutralizing antibodies in vivo after direct immunization of mice against AAV capsid. Results from intramuscular injection of AAV formulated in phosphate-buffered saline into one leg and AAV formulated with MG29 into the other leg showed that MG29 blocked AAV neutralizing antibodies in AAV-immunized mice, allowing for effective gene delivery and AAV re-administration (Figures 19 and 20).

[0273] The second round of rational engineering strategies using Rosetta software involved modifying the affinity of mutant protein M analogs using an average model of many human immunoglobulin structures to predict binding site modifications of mutant protein M analogs that would enhance or decrease affinity and binding. Furthermore, the third round of rational design strategies incorporated the use of both the Rosetta model and the diversity of naturally occurring protein M sequences from different mycoplasma species to create antigenically distinct analogs that do not occur in nature. These analogs can be chimeric, mosaic, or de novo rationally modified amino acid mutants of the whole protein or individual epitopes. The same or different analogs can be used with AAV for multiple rounds of re-administration, even in the presence of inhibitory antibodies raised against the protein M analog. This is because the protein M analog directly competes for antibody binding and blocking antigen recognition (and even its own recognition by inhibitory antibodies). A saturating dose of protein M analog is in excess of the small portion of immunoglobulins raised against the protein M analog after the initial immunization exposure. Furthermore, the generation of protein M analogs with increased epitope diversity could provide an additional layer of immune evasion from protein M-inhibitory antibodies. It is conceivable that each of the listed engineering strategies could be combined or layered on top of each other to generate protein M analogs with multiple distinct properties.

[0274] Finally, we produced a DNA codon-optimized version of Protein M with enhanced protein product yield (Figure 25). Two codon-optimized versions of the parent truncated Protein M sequence were produced: one sequence was optimized for both E. coli and H. sapiens codon usage, and the other was optimized only for H. sapiens codon usage. Protein production in E. coli using the dual E. coli and H. sapiens codon constructs resulted in a four-fold increase in protein yield compared to the parent, non-optimized plasmid. The H. sapiens-only optimized constructs contained IL-2 secretory peptide or albumin secretory peptide sequences, allowing for secretion of the Protein M analog from mammalian cell lines for recovery from the culture medium. Protein M mutant analogs were cloned or synthesized into both types of optimized DNA sequences and used for enhanced protein production. Protein M analogs, stable at 37°C, could be secreted and cultured in human cells without suffering from protein unfolding. Additionally, glycosylation sites on Protein M analogs have been substituted to remove glycosylation at the binding site or to add glycosylation to the outer surface (e.g., analog MG28(N274D)). Addition of outer surface glycosylation sites is another mechanism for enhancing immune evasion of Protein M analogs and for reducing recognition of Protein M-inhibitory antibodies with multiple administrations of Protein M analogs. Finally, Protein M analog DNA sequences have been stably integrated into mammalian cell lines to produce secreted proteins that are stable at 37°C and have either removed or added glycosylation sites.

[0275] method

[0276] AAV virus productionAAV vectors were produced using standard three-plasmid transfection techniques in HEK293 cells. Briefly, the AAV transgene plasmid pTR-CBA-Luc was cotransfected with the AAV Rep / Cap helper plasmid (pXR2 or pXR8) and the adenovirus helper plasmid pXX6-80. After 72 hours, cell cultures were harvested and lysed by freeze-thawing and sonication. The clarified cell lysates were DNAse-treated, ultracentrifuged in a 15% / 25% / 40% / 60% iodixanol step gradient, and purified on an anion-exchange Q-column. The purified AAV vectors were titrated by qPCR using primers designed to amplify segments of the packaged AAV transgene.

[0277] Protein production Plasmid pET-28b(+) encodes protein M analog (a truncated protein from M. genitalium or M. pneumonia lacking the transmembrane domain) and contains an N-terminal His-Tag and a thrombin cleavage site. The plasmid was grown in electrocompetent DH10B cells and purified using the PureLink Maxi-Prep kit from Invitrogen. The pET-28b(+) plasmid was transiently transfected into BL21 / DE3 cells using an overnight starter culture. Autoinduction medium (Magic medium from Invitrogen) was inoculated with the starter culture and grown at 18°C ​​for 3 days. The culture was then pelleted by centrifugation and frozen at -80°C.

[0278] Protein purificationFrozen bacterial cell culture pellets were thawed, lysed by sonication, DNAse-treated, and clarified by centrifugation. The clarified bacterial lysate was dialyzed into nickel binding buffer (20 mM imidazole, 50 mM sodium phosphate (pH 7.4), 500 mM NaCl, 0.02% sodium azide) and passed through a nickel His-trap FF column using FPLC. Protein M bound to the nickel column was then eluted with the same buffer base but containing 500 mM imidazole. Protein M was then passed through an S-100 size-exclusion column, dialyzed into phosphate-buffered saline containing 2% glycerol, and quantified spectrophotometrically. The identity of Protein M was confirmed using SDS-PAGE protein gel electrophoresis for protein separation, followed by Coomassie blue protein staining, which correctly identified the 45-48 kDa Protein M band.

[0279] For the production of some proteins, one of two strategies was implemented to purify Protein M variants. The first protocol (Ni-Purity) involved small-scale production for NanoDSF. Cell pellets were lysed by mixing with a buffer consisting of 2.5 mg / mL lysozyme, 25% B-PER, 75% phosphate-buffered saline (PBS) pH 7.4, and protease inhibitors (PMSF, bestatin, and pepstatin). After 30 min of mixing at room temperature, the crude lysate was centrifuged at 15,000 rcf for 20 min, and the supernatant was incubated with Ni resin for 1 h at 4°C. The resin was then washed (PBS + 20 mM imidazole), and the protein was eluted (PBS + 500 mM imidazole). The eluted protein was exchanged into PBS pH 7.4 using a Zeba desalting column before stability assays. The second protocol (SEC-Purity) was for large-scale production and removed further contaminants / aggregates from the protein sample: cells were lysed via sonication, purified using nickel resin (same buffer as in the first protocol), and finally, samples were frozen after size exclusion chromatography in PBS + 2% glycerol.

[0280] Western blot After protein separation on an 8% SDS-PAGE gel, proteins were transferred onto a PVDF membrane. Immunoblotting was performed using a primary anti-His antibody diluted 1:1000 (10 μg / ml) in 5% nonfat milk. A secondary goat anti-human IgG antibody conjugated to horseradish peroxidase (diluted 1:10,000) was used.

[0281] Determination of protein melting temperature and unfolding Melting temperatures (Tm) were determined using nanodifferential scanning fluorimetry (NanoDSF), and protein unfolding was determined at different temperatures using a circular dichroism assay. The inflection point of the first derivative indicates Tm or unfolding. Proteins were purified according to protocol 1 (Ni-Purity).

[0282] Solubility assay A thermal stability time course was performed by incubating seven aliquots of each SEC-purified protein (0.4 mg / mL) at 37°C for different times. The precipitated protein was pelleted by centrifuging the samples at 15,000 x G for 10 minutes before loading the gel for SDS-PAGE. Proteins were purified according to protocol 2 (SEC-Purity).

[0283] Affinity evaluation by biolayer interferometry Biolayer interferometry was performed at 37°C to assess the affinity of Protein M constructs. Proteins were purified according to Protocol 2 (SEC-Purity). A two-fold dilution series of each M construct ranging from 1000 nM to 15.6 nM was performed using ForteBio Kinetics buffer (PBS + detergent). Binding to an anti-mouse IgG Fc Capture biosensor was assessed. Each protocol included baseline, association, and dissociation steps of 10, 5, and 5 minutes, respectively. Data were subtracted from a reference sensor run in parallel with the association step in buffer. Affinity data were calculated based on a 1:1 curve fit within ForteBio Data Analysis v9.0 software. Reported steady-state binding constants were calculated based on the maximum response at each concentration (n = 7). To minimize X2, kinetic data were only included for the 250 to 15.6 nM dilution range (n = 5).

[0284] Structural Models and Rational Protein EngineeringOptimization of the thermal stability of protein M (PM) analogs was achieved using an in silico rational protein engineering approach based on existing crystal structures of PM bound to immunoglobulins (4NZT and 4NZR). Mutations that stabilize PM peptides were identified using the Rosetta software package. The first protocol involved in silico site-saturation mutagenesis, which repacked amino acid neighbors around the mutated residue. The second protocol involved combinatorial mutations in underpacked regions within the crystal structure. Mutants were ranked according to the difference in score between the wild-type amino acid and the substitution. Further visual inspection identified favorable mutants. Mutants were cloned into the pET-28b+ vector and synthesized by Twist Biosciences. Further design strategies were employed to alter the affinity of the binding site and generate PM analogs with distinct antigenicity.

[0285] A homology model of MP WT was constructed using the Swiss-Model web server (Figure 24). Conservation scores between MG and MP WT isoforms were calculated according to the BLOSUM90 matrix. Images were rendered in PyMol.

[0286] cell culture HEK-293 or Huh7 cells were used for all in vitro AAV neutralization experiments. Cells were grown on 15 cm tissue culture plates and maintained at 37°C in 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin-streptomycin.

[0287] Human IVIG for in vitro neutralization experiments10% human IVIG stock (Gamunex) was purchased from Grifols Therapeutics Inc. (Research Triangle Park, NC, USA). Individual aliquots were diluted to 1 mg / mL in phosphate-buffered saline and stored at -80°C for later use. 12 different mice (50% male, 50% female) were inoculated with 3 × 10 10 After IP administration of the viral genome, AAV8-FVIII, followed by a booster dose of the same vector 2 weeks later and a second boost 6 weeks after the first dose, serum was collected and pooled. Mouse serum was aliquoted and stored at -80°C for future use.

[0288] In vitro AAV neutralization assayNab assays were performed as previously described with minor modifications (Wang et al., Gene Ther. 22:984 (2015)). Cells were pelleted by centrifugation, resuspended in serum-free X-Vivo10 medium, and then plated in 48- or 96-well plate formats. Human IVIG or serum was serially diluted 2-fold or 10-fold. AAV-Luc was incubated with human IVIG or mouse serum for 1 hour at 4°C, followed by AAV addition and an additional 1 hour at 4°C. The AAV + serum incubation was then mixed with cells suspended in serum-free medium at the time of plating. For neutralization experiments using Protein M, three different incubation scenarios were tested (each step for 1 hour at 4°C): neutralizing serum was first incubated with Protein M and then with AAV; AAV was first incubated with neutralizing serum and then with Protein M; and AAV was first incubated with Protein M and then with neutralizing serum. Cells were seeded in 48-well plates in 200 μl medium or 96-well plates in 100 μl medium. After transduction, cells were cultured at 37°C for 24–48 hours to allow expression of the AAV-luciferase transgene. To measure Luc activity, cells were lysed with passive lysis buffer (Promega, Madison, WI, USA), and the luciferase signal was measured using a Wallac 1420 Victor 2 automated plate reader. Nab titer was defined as the highest dilution at which luciferase activity was 50% lower than that of the serum-free control.

[0289] In vivo AAV rechallenge in AAV-immunized miceC57BL / 6 mice were immunized by IP injection of 8E5vg or 1E9vg of AAV8-GFP. Approximately one month later, serum was collected from the mice for titration in an AAV8 in vitro neutralization assay. One day later, IM injections were performed, with each leg containing an equal dose of 1E9vg or 2E9vg AAV8-luciferase, where one leg received AAV formulated in phosphate-buffered saline and the other leg received AAV formulated in a protein M analog, with simple mixing immediately prior to injection. A total volume of 200 μl was injected per leg. Luminescence imaging was performed 2 weeks after AAV-Luc injection after IP administration of D-luciferin substrate.

[0290] The foregoing experiments are illustrative of the present invention and should not be construed as limiting thereof. Although the present invention has been described in detail with reference to preferred embodiments, variations and modifications exist within the scope and spirit of the invention, as described and defined in the following claims.

[0291] JPEG2026000908000005.jpg243166JPEG2026000908000006.jpg243166JPEG2026000908000007.jpg241166JPEG202 6000908000008.jpg244166JPEG2026000908000009.jpg245166JPEG2026000908000010.jpg245166JPEG20260009080 00011.jpg246166JPEG2026000908000012.jpg246166JPEG2026000908000013.jpg242166JPEG2026000908000014.j pg243166JPEG2026000908000015.jpg245166JPEG2026000908000016.jpg246166JPEG2026000908000017.jpg244166

[0292] JPEG2026000908000018.jpg255166JPEG2026000908000019.jpg217140JPEG2026000908000020.jpg218140

[0293] JPEG2026000908000021.jpg247166JPEG2026000908000022.jpg247166

[0294] Table 7: 17 point mutations predicted to improve affinity of MG WT to the antibody. Delta score refers to the score of the mutant minus the score of the WT residue. This list considered 70 residues within 5 Å of the antibody interface in PDB ID: 4NZR. JPEG2026000908000023.jpg136166

[0295] array SEQ ID NO: 1: Wild-type M. genitalium protein M JPEG2026000908000024.jpg232140JPEG2026000908000025.jpg225166

[0296] SEQ ID NO: 2: Soluble form of M. genitalium protein M (amino acid residues 37-556 of SEQ ID NO: 1) with an N-terminal 6-His tag followed by a thrombin cleavage site JPEG2026000908000026.jpg43166JPEG2026000908000027.jpg238140

[0297] SEQ ID NO: 3: Wild-type M. genitalium protein M fragment 74-479 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVASDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPSFSGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDSYPNHFFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLSGNGS QLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFASKPFTHIDLTQVTLQNSDNSAIDANKLKQAVGDIY NYRRFERQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQNGVTFDEN

[0298] SEQ ID NO: 4: Modified M. genitalium protein M MG1 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVASDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPSFSGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDSYPNHTFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLSGNGS QLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFASKPFTHIDLTQVTLQNSDNSAIDANKLKQAVGDIY NYRRFERQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQNGVTFDEN

[0299] SEQ ID NO: 5: Modified M. genitalium protein M MG8 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVASDGSLLSYYQSTEYFNNRVLMPILQ TTNGTLMANNRGYDDVFRQVPSFSGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDQYPNHFFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLSGNGS QLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFASKPFTHIDLTQVTLQNSDNSAIDANKLKQAVGDIY NYRRFERQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQNGVTFDEN

[0300] SEQ ID NO: 6: Modified M. genitalium protein M MG13 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVASDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPSFSGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDSYPNHFFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLSGNGS QLNELDLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFASKPFTHIDLTQVTLQNSDNSAIDANKLKQAVGDIYNYRRFERQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQNGVTFDEN

[0301] SEQ ID NO: 7: Modified M. genitalium protein M MG15 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVAEDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPRFPGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDSYPNHFFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLSGNGS QLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFASKPFTHIDLTQVTLQNSDNSAIDANKLKQAVGDIY NYRRFERQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQNGVTFDEN

[0302] SEQ ID NO: 8: Modified M. genitalium protein M MG21 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVASDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPSFSGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDSYPNHFFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLSGNGS QLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFASKPFTHIDLTQVTLQNSDNSAIDANKLKQAVGDIYNYRRFERQFQGYFAGGYIDKYLIKIVNTNPDVDDDIVYRSLKELNLHLEEAYREGDNIYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQNGVTFDEN

[0303] SEQ ID NO: 9: Modified M. genitalium protein M MG22 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVASDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPSFSGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDSYPNHFFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLSGNGS QLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFASKPFTHIDLTQVTLQNSDNSAIDANKLKQAIGDIYNYRRFERQFQGYFAGGYIDKYLIKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQNGVTFDEN

[0304] SEQ ID NO: 10: Modified M. genitalium protein M MG23 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVASDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPSFSGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDSYPNHFFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLSGNGS QLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFASKPFTHIDLTQVTLQNSDNSAIDANKLKQAVGDIYNYRRFERQFQGYFAGGYIDKYLVKLVNTNPDVDDDIVYRSLKELNLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQNGVTFDEN

[0305] SEQ ID NO: 11: Modified M. genitalium protein M MG24 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVASDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPSFSGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDSYPNHFFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLSGNGS QLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFVSKPFTHIDLTQVTLQNSDNSAIDANKLKQAVGDIY NYRRFERQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQNGVTFDEN

[0306] SEQ ID NO: 12: Modified M. genitalium protein M MG27 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVAEDGSLLSYYQSTEYFNNRVLMPILQ TTNGTLMANNRGYDDVFRQVPRFPGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDQYPNHTFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLSGNGS QLNELDLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFVSKPFTHIDLTQVTLQNSDNSAIDANKLKQAIGDIYNYRRFERQFQGYFAGGYIDKYLIKIVNTNPDVDDDIVYRSLKELNLHLEEAYREGDNIYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQNGVTFDEN

[0307] SEQ ID NO: 13: Modified M. genitalium protein M MG28 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVASDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPSFSGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDSYPNHFFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLSGDGS QLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFASKPFTHIDLTQVTLQNSDNSAIDANKLKQAVGDIY NYRRFERQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQNGVTFDEN

[0308] SEQ ID NO: 14: Modified M. genitalium protein M MG29 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVAEDGSLLSYYQSTEYFNNRVLMPILQ TTNGTLMANNRGYDDVFRQVPRFPGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDQYPNHTFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLSGNGS QLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFVSKPFTHIDLTQVTLQNSDNSAIDANKLKQAVGDIY NYRRFERQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQNGVTFDEN

[0309] SEQ ID NO: 15: Modified M. genitalium protein M MG31 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVASDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPSFSGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDSYPNHFFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLSGNGS QLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFASKPFTHIDLTQVTLQNSDNSAIDANKLKQAIGDIYNYRRFERQFQGYFAGGYIDKYLIKIVNTNPDVDDDIVYRSLKELNLHLEEAYREGDNIYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQNGVTFDEN

[0310] SEQ ID NO: 16: Modified M. genitalium protein M MG33 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVASDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPSFSGWSNTKPTTVSTSNNLTYDKWTYFAAKGSPLYDSYPNHFFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLSGNGS QLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFASKPFTHIDLTQVTLQNSDNSAIDANKLKQAVGDIY NYRRFERQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQNGVTFDEN

[0311] SEQ ID NO: 17: Modified M. genitalium protein M MG38 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVASDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPSFSGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDSYPNHFFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLSGNGS QLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFASKPFTHIDLTQVDLQNSDNSAIDANKLKQAVGDIY NYRRFERQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQNGVTFDEN

[0312] SEQ ID NO: 18: Modified M. genitalium protein M MG40 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVASDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPSFSGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDSYPNHFFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLSGNGS QLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFASKPFTHIDLTQVPLQNSDNSAIDANKLKQAVGDIY NYRRFERQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQNGVTFDEN

[0313] SEQ ID NO: 19: Modified M. genitalium protein M MG43 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVAEDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPRFPGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDSYPNHFFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLSGNGS QLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFVSKPFTHIDLTQVTLQNSDNSAIDANKLKQAIGDIYNYRRFERQFQGYFAGGYIDKYLIKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQNGVTFDEN

[0314] SEQ ID NO: 20: Modified M. genitalium protein M MG44 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVAEDGSLLSYYQSTEYFNNRVLMPILQ TTNGTLMANNRGYDDVFRQVPRFPGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDQYPNHTFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLSGNGS QLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFVSKPFTHIDLTQVTLQNSDNSAIDANKLKQAIGDIYNYRRFERQFQGYFAGGYIDKYLIKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQNGVTFDEN

[0315] SEQ ID NO: 21: Modified M. genitalium protein M MG45 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVASDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPSFSGWCNTKATTVSTSNNLTYDKWTYFACKGSPLYDSYPNHFFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLSGNGS QLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFASKPFTHIDLTQVTLQNSDNSAIDANKLKQAVGDIY NYRRFERQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQNGVTFDEN

[0316] SEQ ID NO: 22: Modified M. genitalium protein M MG46 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVAEDGSLLSYYQSTEYFNNRVLMPILQ TTNGTLMANNRGYDDVFRQVPRFPGWCNTKATTVSTSNNLTYDKWTYFACKGSPLYDQYPNHTFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLSGNGS QLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFVSKPFTHIDLTQVTLQNSDNSAIDANKLKQAVGDIY NYRRFERQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQNGVTFDEN

[0317] SEQ ID NO: 23: Wild-type M. pneumoniae protein M MKLNFKIKDKKTLKRLKKGGFWALGLFGAAINAFSAVLIVNEVLRLQSGETLIASGRSGNLSFQLYSKVNQNAKSKLNSISLTDGYRSEIDLGDGSNFREDFRNFANNLSEAITDAPKDLLRPVPKVEVSGLIKTSSTFITPNF KAGYYDQVAADGKTLKYYQSTEYFNNRVVMPILQTTNGTLTANNRAYDDIFVDQGVPKFPGWFHDVDKAYYAGSNGQSEYLFKEWNYYVANGSPLYNVYPNHHFKQIKTIAFDAPRIKQGNTDGINLNLKQRNPDYVIINGLTGDG STLKDLELPESVKKVSIYGDYHSINVAKQIFKNVLELEFYSTNQDNNFGFNPLVLGDHTNIIYDLFASKPFNYIDLTSLELKDNQDNIDASKLKRAVSDIYIRRRFERQMQGYWAGGYIDRYLVKNTNEKNVNKDNDTVYAALKD INLHLEETYTHGGNTMYRVNENYYPGASAYEAERATRDSEFQKEIVQRAELIGVVFEYGVKNLRPGLKYTVKFESPQEQVALKSTDKFQPVIGSVTDMSKSVTDLIGVLRDNAEILNITNVSKDETVVAELKEKLDRENVFQEIRT

[0318] SEQ ID NO: 24: Wild-type M. pneumoniae protein M fragment SISLTDGYRSEIDLGDGSNFREDFRNFANNLSEAITDAPKDLLRPVPKVEVSGLIKTSSTFITPNFKAGYYDQVAADGKTLKYYQSTEYFNNRVVMPILQTT NGTLTANNRAYDDIFVDQGVPKFPGWFHDVDKAYYAGSNGQSEYLFKEWNYYVANGSPLYNVYPNHHFKQIKTIAFDAPRIKQGNTDGINLNLKQRNPDYVIIN GLTGDGSTLKDLELPESVKKVSIYGDYHSINVAKQIFKNVLELEFYSTNQDNNFGFNPLVLGDHTNIIYDLFASKPFNYIDLTSLELKDNQDNIDASKLKRAVS DIYIRRRFERQMQGYWAGGYIDRYLVKNTNEKNVNKDNDTVYAALKDINLHLEETYTHGGNTMYRVNENYYPGASAYEAERATRDSEFQKEIVQRAELIGVVFE

[0319] SEQ ID NO: 25: Polynucleotide encoding M. genitalium protein M, codon-optimized for both bacterial and human expression

[0320] SEQ ID NO: 26: Polynucleotide encoding M. genitalium protein M codon-optimized for human expression

[0321] SEQ ID NO: 27: Modified M. genitalium protein M MG47 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVAEDGSLLSYYQSTEYFNNRVLMPILQ TTNGTLMANNRGYDDVFRQVPRFPGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDQYPNHTFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLSGNGS QLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFVSKPFTHIDLTQVTLQNSDNSAIDANKLKQAVGDIY NYRRFERQFQGYEAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYKPGASIYENERASRDSEFQNEILKRAEQNGVTFDEN

[0322] SEQ ID NO: 28: Modified M. genitalium protein M MG48 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVAEDGSLLSYYQSTEYFNNRVLMPILQ TTNGTLMANNRGYDDVFRQVPRFPGWCNTKPTTVSTSNNLTYDKWTYFACKGSPLYDQYPNHTFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLSGDGS QLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFVSKPFTHIDLTQVPLQNSDNSAIDANKLKQAVGDIY NYRRFERQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQNGVTFDEN

[0323] SEQ ID NO: 29: Modified M. genitalium protein M MG49 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVAEDGSLLSYYQSTEYFNNRVLMPILQ TTNGTLMANNRGYDDVFRQVPRFPGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDQYPNHTFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGLSGNGS QLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFVSKPFTHIDLTQVTLQNSDNSAIDANKLKQAVGDIY NYRRFERQFQGYFPGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQNGVTFDEN

[0324] SEQ ID NO: 30: Modified M. genitalium protein M MP29 SISLTDGYRSEIDLGDGSNFREDFRNFANNLSEAITDAPKDLLRPVPKVEVSGLIKTSSTFITPNFKAGYYDQVAEDGKTLKYYQSTEYFNNRVVMPILQ TTNGTLTANNRAYDDIFVDQGVPRFPGWFHDVDKAYYAGSNGQSEYLFKEWNYYVANGSPLYNQYPNHTFKQIKTIAFDAPRIKQGNTDGINLNLKQRNPDY VIINGLTGDGSTLKDLELPESVKKVSIYGDYHSINVAKQIFKNVLELEFYSTNQDNNFGFNPLVLGDHTNIIYDLFVSKPFNYIDLTSLELKDNQDNIDASKLKRAVSDIYIRRRFERQMQGYWAGGYIDRYLVKNTNEKNVNKDNDTVYAALKDINLHLEETYTHGGNTMYRVNENYYPGASAYEAERATRDSEFQKEIVQR

Claims

1. A modified Mycoplasma protein M or a functional fragment thereof, having one or more amino acid mutations that increase or maintain the thermal stability of Mycoplasma protein M or a functional fragment thereof compared to wild-type Mycoplasma protein M or a functional fragment thereof; A modified Mycoplasma protein M or a functional fragment thereof.

2. 2. The modified Mycoplasma protein M or a functional fragment thereof according to claim 1, having one or more amino acid mutations that increase the thermal stability of Mycoplasma protein M or a functional fragment thereof compared to wild-type Mycoplasma protein M or a functional fragment thereof; A modified Mycoplasma protein M or a functional fragment thereof.

3. 3. A modified Mycoplasma protein M or a functional fragment thereof according to claim 1 or 2, derived from protein M of Mycoplasma genitalium or Mycoplasma pneumoniae, A modified Mycoplasma protein M or a functional fragment thereof.

4. 4. A modified Mycoplasma protein M or a functional fragment thereof according to any one of claims 1 to 3, a fragment of about residue 74 to about residue 479 of M. genitalium protein M (SEQ ID NO:3) or the corresponding residues of M. pneumoniae protein M (SEQ ID NO:23); A modified Mycoplasma protein M or a functional fragment thereof.

5. The one or more mutations are located within 5 Å of the antibody binding site of Protein M (residues 95, 99, 102, 103, 105, 106, 107, 109, 110, 114, 116, 117, 118, 119, 120, 144, 158, 160, 161, 162, 163, 177, 178, 179, 180, 181, 186, 187, 188, 191, 321, 33 8, 340, 341, 345, 381, 384, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 426, 427, 429, 436, 438, 439, 440, 441, 442, 444, 445, 446, 447, 448, 449, 452, 453, 455, 456, 457, 462, 466), or at any of residues 469-479 of M. genitalium protein M (SEQ ID NO: 1), or at the corresponding residues of M. pneumoniae protein M (SEQ ID NO: 23), A modified Mycoplasma protein M or a functional fragment thereof according to any one of claims 1 to 4.

6. The one or more mutations are selected from the group consisting of residues 78, 81, 83, 84, 85, 89, 90, 91, 92, 93, 94, 96, 97, 100, 101, 108, 111, 112, 113, 122, 123, 125, 126, 127, 128, 130, 131, 133, 134, 136, 137, 139, 141, 142, 146, 147, 148, 149, 150, 153, 154, 155, 156, 164, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223 , 170, 175, 176, 184, 185, 189, 192, 193, 196, 198, 201, 202, 204, 205, 206, 207, 209, 211, 215, 218, 220, 224, 225, 226, 227, 231, 232, 234, 235, 236, 237, 239, 241, 243, 244, 245, 246, 247, 249, 250, 252, 253, 254, 255, 256, 257, 258, 259, 264, 269, 270, 272, 274, 275, 276, 279, 282, 284, 286, 287, 288, 291, 297, 299, 300, 302, 303, 304, 305, 307, 308, 309, 310, 311, 313, 317, 318, 319, 320, 322, 326, 327, 329, 331, 332, 333, 335, 337, 342, 343, 347, 348, 351, 354, 355, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 58, 359, 360, 361, 362, 363, 367, 369, 370, 371, 372, 373, 374, 375, 378, 385, 399, 400, 401, 402, 405, 406, 407, 408, 409, 411, 413, 414, 417, 418, 419, 424, 428, 434, 435, 443, 450, 459, 460, 463, 464, 465, 468, or any combination thereof, or at the corresponding residues of M. pneumoniae protein M (SEQ ID NO: 23); A modified Mycoplasma protein M or a functional fragment thereof according to any one of claims 1 to 5.

7. 7. The modified Mycoplasma protein M or functional fragment thereof of claim 6, wherein the one or more mutations are those listed in Table 4 or any combination thereof.

8. The one or more mutations are selected from the group consisting of residues 83, 90, 92, 94, 137, 142, 147, 150, 156, 184, 196, 198, 205, 211, 215, 225, 231, 232, 234, 235, 236, 237, 239, 243, 245, 250, 255, 256, 259, 264, 272, 274, 276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 339, 343, 345, 346, 347, 348, 349, 350, 355, 356, 6. The modified Mycoplasma protein M or functional fragment thereof of any one of claims 1 to 5, wherein the amino acid sequence is at residues 5, 276, 279, 282, 297, 300, 302, 310, 320, 326, 331, 332, 335, 342, 343, 347, 348, 355, 357, 361, 371, 374, 378, 385, 401, 402, 409, 413, 424, 460, 463, 464, 468, or any combination thereof, or the corresponding residues of M. pneumoniae protein M (SEQ ID NO: 23).

9. 9. The modified Mycoplasma protein M or functional fragment thereof of claim 8, wherein the one or more mutations are those listed in Table 5 or any combination thereof.

10. 6. The modified Mycoplasma protein M or functional fragment thereof of any one of claims 1 to 5, wherein the one or more mutations are at residues 150, 196, 198, 201, 205, 224, 232, 237, 274, 282, 342, 355, 373, 400, 402, 407, 409, 413, 135, or any combination thereof, of M. genitalium protein M (SEQ ID NO: 1), or the corresponding residues of M. pneumoniae protein M (SEQ ID NO: 23).

11. the one or more mutations are M. genitalium protein M (SEQ ID NO: 1), a) 237; b) 232; c) 282; d) 150, 196, 198, 400, 402, 407, 409; e) 413, 435; f) 373, 400; g) 402, 407, 409, 413; h) 342; i) 150, 196, 198, 232, 237, 282, 342, 373, 400, 402, 407, 409, 413, 435; j) 274; k) 150, 196, 198, 232, 237, 342, 400, 402, 407, 409; l) 373, 413, 435; m) 205; n) 355; o) 150, 196, 198, 342, 373, 400, 402, 407, 409; p) 150, 196, 198, 232, 237, 342, 373, 400, 402, 407, 409; q) 201, 224; r) 150, 196, 198, 201, 224, 232, 237, 342, 400, 402, 407, 409; s) 150, 196, 198, 232, 237, 342, 390, 400, 402, 407, 409, 444; t) 150, 196, 198, 201, 205, 224, 232, 237, 274, 342, 355, 400, 402, 407, 409; or u) 150, 196, 198, 232, 237, 342, 391, 400, 402, 407, 409 a residue selected from or at the corresponding residues of M. pneumoniae protein M (SEQ ID NO: 23): The modified Mycoplasma protein M or a functional fragment thereof according to claim 10.

12. the one or more mutations are M. genitalium protein M (SEQ ID NO: 1), a) F237T; b) S232Q; c) Q282D; d) S150E, S196R, S198P, V400I, N402I, K407P, S409V; e) L413I, T435I; f) V373I, V400I; g) N402L, K407P, S409V, L413I; h) A342V; i) S150E, S196R, S198P, S232Q, F237T, Q282D, A342V, V373I, V400I, N402I, K407P, S409V, L413I, T435I; j) N274D; k) S150E, S196R, S198P, S232Q, F237T, A342V, V400I, N402I, K407P, S409V; l) V373I, L413I, T435I; m) A205P; n) T355D; o) T355P; p) S150E, S196R, S198P, A342V, V373I, V400I, N402I, K407P, S409V; q) 150, 196, 198, 232, 237, 342, 373, 400, 402, 407, 409; r) S201C, A224C; s) S150E, S196R, S198P, S201C, A224C, S232Q, F237T, A342V, V400I, N402I, K407P, S409V; t) S150E, S196R, S198P, S232Q, F237T, A342V, F390E, V400I, N402I, K407P, S409V Y444K; u) S150E, S196R, S198P, S201C, A205P, A224C, S232Q, F237T, N274D, A342V, T355P, V400I, N402I, K407P, S409V; or v) S150E, S196R, S198P, S232Q, F237T, A342V, A391P, V400I, N402I, K407P, S409V or the corresponding residues of M. pneumoniae protein M (SEQ ID NO: 23) 12. The modified Mycoplasma protein M or a functional fragment thereof according to claim 11, selected from:

13. 6. The modified Mycoplasma protein M or functional fragment thereof of any one of claims 1 to 5, wherein the one or more mutations are at residues 155, 203, 243, 248, and 358 of M. pneumoniae protein M (SEQ ID NO: 23).

14. 14. The modified Mycoplasma protein M or functional fragment thereof of claim 13, wherein the one or more mutations are A155E, K203R, H243T, V248Q, and A358V of M. pneumoniae protein M (SEQ ID NO: 23).

15. the one or more mutations are M. genitalium protein M (SEQ ID NO: 1), a) 468; b) 150; c) 147; d) 272; e) 355; f) 276, 277, 279; g) 300; h) 378; i) 156; j) 232; k) 245; l) 276; m) 225; or n) 310 a residue selected from or the corresponding residues of M. pneumoniae protein M (SEQ ID NO: 23), or a functional fragment thereof, of any one of claims 1 to 5.

16. the one or more mutations are M. genitalium protein M (SEQ ID NO: 1), a) R468Q; b) S150E; c) H147F; d) S272G; e) T355G; f) S276E, Q277L, N279R; g) N300Q; h) N378Y; i) S156K; j) S232L; k) A245Q; l) S276D; m) K225P; or n) V310E or the corresponding residues of M. pneumoniae protein M (SEQ ID NO: 23) 16. The modified Mycoplasma protein M or a functional fragment thereof according to claim 15, selected from:

17. 17. A modified Mycoplasma protein M or a functional fragment thereof according to any one of claims 1 to 16, wherein the modified Mycoplasma protein M or a functional fragment thereof is further modified to remove one or more glycosylation sites.

18. 17. A modified Mycoplasma protein M or a functional fragment thereof according to any one of claims 1 to 16, wherein the modified Mycoplasma protein M or a functional fragment thereof is further modified to add one or more glycosylation sites.

19. A modified Mycoplasma protein M or a functional fragment thereof according to any one of claims 1 to 4, further comprising one or more mutations that increase the affinity of the modified Mycoplasma protein M or a functional fragment thereof for an antibody.

20. 20. The modified Mycoplasma protein M or functional fragment thereof of claim 19, wherein the one or more mutations are at residues 95, 102, 103, 106, 107, 114, 116, 160, 161, 162, 163, 181, 186, 321, 381, 384, 389, 390, 391, 396, 397, 426, 429, 436, 438, 439, 441, 442, 447, 448, 449, 452, 453, 455, 456, 462, or 466 of M. genitalium protein M (SEQ ID NO: 1), or any combination thereof, or the corresponding residues of M. pneumoniae protein M (SEQ ID NO: 23).

21. 21. The modified Mycoplasma protein M or functional fragment thereof of claim 20, wherein the one or more mutations are those listed in Table 6 or any combination thereof.

22. 20. The modified Mycoplasma protein M or functional fragment thereof of claim 19, wherein the one or more mutations are at residues 95, 103, 116, 186, 321, 389, 429, 442, or 466 of M. genitalium protein M (SEQ ID NO: 1), or any combination thereof, or the corresponding residues of M. pneumoniae protein M (SEQ ID NO: 23).

23. 23. The modified Mycoplasma protein M or functional fragment thereof of claim 22, wherein the one or more mutations are those listed in Table 7 or any combination thereof.

24. A modified Mycoplasma protein M or a functional fragment thereof according to any one of claims 1 to 4, further comprising one or more mutations that reduce the affinity of the modified Mycoplasma protein M or a functional fragment thereof for an antibody.

25. 20. The modified Mycoplasma protein M or functional fragment thereof of claim 19, wherein the one or more mutations are at residues 390 and / or 444 of M. genitalium protein M (SEQ ID NO: 1) or the corresponding residues of M. pneumoniae protein M (SEQ ID NO: 23).

26. 26. The modified Mycoplasma protein M or a functional fragment thereof according to claim 25, wherein said one or more mutations are 390E and / or Y444K.

27. 27. A modified Mycoplasma protein M or a functional fragment thereof according to any one of claims 1 to 26, further comprising a secretory peptide at the N-terminus.

28. 28. A polynucleotide encoding a modified Mycoplasma protein M or a functional fragment thereof according to any one of claims 1 to 27.

29. 29. The polynucleotide of claim 28 operably linked to a promoter.

30. 30. The polynucleotide of claim 29, wherein the promoter is a bacterial promoter.

31. 30. The polynucleotide of claim 29, wherein the promoter is a mammalian promoter.

32. 32. The polynucleotide of any one of claims 28 to 31, wherein the polynucleotide is codon-optimized for expression in a bacterium, such as E. coli.

33. 32. The polynucleotide of any one of claims 28 to 31, wherein the polynucleotide is codon-optimized for expression in a mammalian cell, such as a human cell.

34. 32. The polynucleotide of any one of claims 28 to 31, wherein the polynucleotide is codon-optimized for expression in both bacteria, such as E. coli, and mammalian cells, such as human cells.

35. A vector comprising the polynucleotide of any one of claims 28 to 34.

36. 36. The vector of claim 35, wherein the vector is a bacterial vector.

37. 36. The vector of claim 35, wherein the vector is a mammalian vector.

38. A transformed cell comprising a polynucleotide according to any one of claims 28 to 34 and / or a vector according to any one of claims 35 to 37.

39. 39. The transformed cell of claim 38, which is a bacterial cell such as E. coli.

40. 39. The transformed cell of claim 38, which is a mammalian cell, such as a human cell.

41. 41. The transformed cell of any one of claims 38 to 40, wherein the polynucleotide is stably integrated into the genome of the cell.

42. A method for inhibiting neutralization of a heterologous drug by a neutralizing antibody when the heterologous drug is administered to a subject, comprising: administering to said subject an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, thereby inhibiting neutralization of said heterologous agent. method.

43. 43. The method of claim 42, wherein the heterologous agent is a nucleic acid delivery vector.

44. 44. The method of claim 43, wherein the nucleic acid delivery vector is a viral vector.

45. 45. The method of claim 44, wherein the viral vector is an adeno-associated virus, retrovirus, lentivirus, poxvirus, alphavirus, baculovirus, vaccinia virus, herpesvirus, Epstein-Barr virus, or adenovirus vector.

46. 44. The method of claim 43, wherein the nucleic acid delivery vector is a non-viral vector.

47. 47. The method of claim 46, wherein the non-viral vector is a plasmid, a liposome, a charged lipid, a nucleic acid-protein complex, or a biopolymer.

48. 43. The method of claim 42, wherein the heterologous agent is a gene editing complex.

49. 49. The method of Claim 48, wherein the gene editing complex is a CRISPR complex.

50. 43. The method of claim 42, wherein the heterologous agent is a protein or a nucleic acid.

51. 51. The method of claim 50, wherein the protein is an enzyme.

52. 51. The method of claim 50, wherein the nucleic acid is an antisense nucleic acid or an inhibitory RNA.

53. 53. The method of any one of claims 42 to 52, comprising: The effective amount of Protein M is an amount sufficient to inhibit neutralization by at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9%. method.

54. 54. The method of any one of claims 42 to 53, comprising: The effective amount of Protein M is sufficient to provide a ratio of Protein M to total immunoglobulin in the subject on a molar basis of about 0.5:1 to about 8:1, e.g., about 2:

1. method.

55. 55. The method of any one of claims 42 to 54, wherein the protein M or a functional fragment or derivative thereof is administered to the subject prior to administration of the heterologous agent.

56. 55. The method of any one of claims 42 to 54, wherein said Protein M or a functional fragment or derivative thereof is administered to said subject simultaneously with administration of said heterologous agent.

57. 57. The method of claim 56, wherein the heterologous agent is combined with the protein M or a fragment or derivative thereof prior to administration to the subject.

58. 58. The method of any one of claims 42 to 57, wherein the protein M or a functional fragment or derivative thereof is derived from Mycoplasma genitalium, Mycoplasma pneumoniae, or Mycoplasma penetrans.

59. 59. The method of any one of claims 42 to 58, wherein the Protein M or functional fragment or derivative thereof is a Protein M fragment.

60. 60. The method of claim 59, wherein the functional fragment of protein M comprises amino acid residues 17-537, 37-556, 37-482, 37-468, 37-442, 74-468, 74-479, 74-482, 74-468, 74-442, or 74-556 of M. genitalium protein M (SEQ ID NO: 1), or the corresponding residues from another protein M.

61. 61. The method of any one of claims 42 to 60, wherein said protein M or a functional fragment or derivative thereof is a modified Mycoplasma protein M or a functional fragment thereof according to any one of claims 1 to 24.

62. 62. The method of any one of claims 42 to 61, wherein the Protein M or a functional fragment or derivative thereof is administered to the subject more than once.

63. 63. The method of claim 62, wherein said protein M or a functional fragment or derivative thereof is administered to said subject each time said heterologous agent is administered to said subject.

64. 64. The method of claim 62 or 63, wherein the same protein M or a functional fragment or derivative thereof is administered each time.

65. 64. The method of claim 62 or 63, wherein a different protein M or functional fragment or derivative thereof is administered each time.

66. 66. The method of any one of claims 42 to 65, further comprising administering to the subject an additional treatment to reduce antibody concentration in the subject.

67. 67. The method of claim 66, wherein the additional treatment is plasma exchange, administration of an antibody-digesting enzyme such as IdeS or IdeZ, splenectomy, chemotherapy, immunotherapy, or radiation therapy.

68. 68. The method of claim 66 or 67, wherein the additional treatment is administered before, during, and / or after administration of protein M or a functional fragment or derivative thereof.

69. 1. A method for expressing a polypeptide or functional nucleic acid in a subject, comprising: administering to the subject (a) a nucleic acid delivery vector encoding the polypeptide or functional nucleic acid, and (b) an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof; Thereby, the polypeptide or functional nucleic acid is expressed in the subject. method.

70. 1. A method of treating a disorder in a subject in need thereof, comprising: the disorder is treatable by expressing a polypeptide or functional nucleic acid in the subject; administering to the subject (a) a therapeutically effective amount of a nucleic acid delivery vector encoding the polypeptide or functional nucleic acid, and (b) an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof; thereby treating said disorder in said subject. method.

71. 1. A method of editing a gene in a subject, comprising: administering to the subject (a) an effective amount of a gene editing complex, and (b) an effective amount of Mycoplasma protein M, or a functional fragment or derivative thereof; thereby expressing the polypeptide or functional nucleic acid in said subject. method.

72. 1. A method of treating an autoimmune disease in a subject in need thereof, comprising: administering to said subject a therapeutically effective amount of Mycoplasma protein M or a functional fragment or derivative thereof; thereby treating said autoimmune disease. method.

73. 1. A method for treating a disorder associated with excess antibodies in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, thereby treating said disorder associated with excess antibodies. method.

74. 74. The method of claim 73, wherein the disorder associated with excess antibodies is multiple myeloma, monoclonal gammopathy of undetermined significance (MGUS), or Waldenstrom's macroglobulinemia.

75. 1. A method for acutely inhibiting antibodies in a subject in need thereof, comprising: administering to said subject a therapeutically effective amount of Mycoplasma protein M or a functional fragment or derivative thereof; thereby acutely inhibiting antibodies, method.

76. 76. The method of claim 75, wherein the subject has cytokine release syndrome or an acute autoimmune attack, e.g., sudden-onset severe autoimmune vasculitis.

77. 77. A method according to any one of claims 69 to 76, wherein the protein M or a functional fragment or derivative thereof is a modified Mycoplasma protein M or a functional fragment thereof according to any one of claims 1 to 27.

78. 78. The method of any one of claims 42 to 77, wherein the Mycoplasma protein M or functional fragment or derivative thereof is administered to the subject by a route selected from oral, rectal, transmucosal, intranasal, inhalation, buccal (e.g. sublingual), vaginal, intrathecal, intraocular, intravitreal, intracochlear, transdermal, intraendothelial, intrauterine (or intraembryonic), parenteral (e.g. intravenous, subcutaneous, intradermal, intracranial, intramuscular (including administration to skeletal muscle, diaphragm and / or cardiac muscle), intrapleural, intracerebral, and intraarticular), topical (e.g. both skin and mucosal surfaces (including respiratory tract surfaces), and transdermal administration), intralymphatic, etc., and direct tissue or organ injection (e.g. liver, eye, skeletal muscle, cardiac muscle, diaphragm muscle, or brain).

79. 78. The method of any one of claims 42 to 77, wherein the Mycoplasma protein M or a functional fragment or derivative thereof is administered to skeletal muscle, smooth muscle, heart, diaphragm, respiratory epithelium, liver, kidney, spleen, pancreas, skin, lung, eye, or ear.

80. 80. The method of any one of claims 42 to 79, wherein the Mycoplasma protein M or a functional fragment or derivative thereof is administered to a diseased tissue or organ.

81. 81. The method of any one of claims 42 to 80, wherein the subject is a human.

82. 1. A method for isolating a compound comprising an antibody light chain variable region and / or heavy chain variable region from a sample, comprising:

28. A method for producing a compound comprising contacting said compound with a modified Mycoplasma protein M or functional fragment thereof according to any one of claims 1 to 27 attached to a solid support, and then eluting said compound from said modified Mycoplasma protein M or functional fragment thereof. method.

83. 83. The method of claim 82, wherein the compound comprising an antibody light chain variable region and / or heavy chain variable region is an antibody or an antigen-binding fragment thereof.

84. 84. The method of claim 82 or 83, wherein the solid support is a bead or particle.

85. 85. The method of any one of claims 82 to 84, wherein the solid support comprises agarose, polyacrylamide, dextran, cellulose, polysaccharides, nitrocellulose, silica, alumina, aluminum oxide, titania, titanium oxide, zirconia, styrene, polyvinyl difluoride nylon, copolymers of styrene and divinylbenzene, polymethacrylate esters, derivatized azlactone polymers or copolymers, glass, or cellulose.

86. 86. The method of any one of claims 82 to 85, wherein the compound is eluted by a change in pH.

87. 28. A modified Mycoplasma protein M or a functional fragment thereof according to any one of claims 1 to 27 attached to a solid support.

88. 1. A method of performing an immunoassay, comprising:

28. A method for the preparation of a modified Mycoplasma protein M or a functional fragment thereof according to claim 1, wherein the modified Mycoplasma protein M or a functional fragment thereof is used to bind a compound comprising an antibody light chain variable region and / or a heavy chain variable region. method.

89. 89. The method of claim 88, wherein the immunoassay is selected from a radioimmunoassay (RIA), an enzyme-linked immunosorbent assay (ELISA) assay, an enzyme-linked immunosorbent assay (EIA), a sandwich assay, a gel diffusion precipitation reaction, an immunodiffusion assay, an agglutination assay, an immunofluorescence assay, a fluorescence-activated cell sorting (FACS) assay, an immunohistochemistry assay, a protein A immunoassay, a protein G immunoassay, a protein L immunoassay, a biotin / avidin assay, a biotin / streptavidin assay, an immunoelectrophoresis assay, a precipitation / flocculation reaction, an immunoblot (Western blot; dot / slot blot); an immunodiffusion assay; a liposome immunoassay, a chemiluminescence assay, a library screening, an expression array, an immunoprecipitation, a competitive binding assay, and an immunohistochemical staining.

90. 90. A kit comprising the modified Mycoplasma protein M or functional fragment thereof of any one of claims 1 to 27 or 87.

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