Protein M analogs and fusion proteins and their use to inhibit antibody function - Patents.com

Abstract

The present invention relates to methods and compositions for binding 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 an 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 thermal stability compared to wild-type protein M, and their use in the methods of the present invention.

Description

[Technical field]

[0001] Priority statement This application claims the benefit of U.S. Provisional Application No. 63 / 145,188, filed February 3, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates to methods and compositions for binding 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 an 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 thermal stability 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 to correct genetic diseases. Among gene therapies, the delivery vehicle, adeno-associated virus (AAV) vectors, has shown therapeutic efficacy in numerous clinical trials. The first FDA-approved gene therapy was used in blind patients. The number of clinical trials on 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 of target tissues. More than 90% of the general population has been exposed to AAV via 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] To overcome AAV NAbs, several approaches have been employed: serial plasma exchange, immunosuppressive drugs, and vector capsid modification to remove immune epitopes. Plasma exchange is inefficient, requiring multiple rounds to remove 2-3x the remaining NAbs per session, and only low titers of NAbs can be addressed. Plasma exchange is also time consuming and exposes patients to the risk of nosocomial infection transmission through concurrent antibody depletion and repeated intravenous needle exposure. Steroids or pharmacological immunosuppression to kill B cells have significant health risks and require long-term regimens to further reduce NAbs by 10-fold. AAV capsid engineering is an innovative method, but is ultimately insufficient against polyclonal anti-AAV sera, with only a 10-fold increase in antibody escape observed in vivo. Furthermore, modification of 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 that exceed 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 capable of non-specifically binding antibodies and blocking the ability of antibodies to bind antigens (U.S. Patent No. 9,593,150; U.S. Publication No. 2017 / 0320921).

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

[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 the method is effective over a wide range of existing NAb concentrations. The present invention exploits the use of a unique mycoplasma-derived protein and its analogs (called protein M) to enable successful gene delivery by preventing neutralization of heterologous agents (e.g., AAV vectors) by NAbs when the heterologous agents are 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 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 escaping from NAb was shown to be proportional to NAb titer and volume, amount of protein M, and 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 prior to AAV administration or formulated with AAV for NAb escape. The efficacy of this approach depends on the interaction of protein M with immunoglobulins before AAV is neutralized. This approach can be used to overcome NAb against multiple heterologous 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 in which it is beneficial to bind antibodies, 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 M proteins having increased thermal stability and / or other advantageous properties (e.g., increased or maintained pH stability compared to wild-type Mycoplasma protein M or functional fragments thereof) for carrying out the methods of the present invention in vivo or at elevated temperatures.

[0010] Thus, one aspect of the invention relates to modified Mycoplasma protein M or a functional fragment thereof having one or more amino acid mutations that increase or maintain the thermal stability and / or pH stability of Mycoplasma protein M or a functional fragment thereof compared to wild-type Mycoplasma protein M or a functional fragment thereof. In some embodiments, the thermal stability is maintained up to 75° C., 70° C., 65° C., 60° C., 55° C., 50° C., 45° C., 40° C., or 38° C., and / or the pH stability is maintained at pH 2-8, pH 2.5-7.5, or pH 4.5-7.5.

[0011] Another aspect of the present invention relates to a fusion protein comprising a modified Mycoplasmal protein M or a functional fragment thereof, a linker and a peptide.

[0012] A further aspect of the present invention relates to a polynucleotide encoding the modified Mycoplasma protein M of the present invention or a functional fragment thereof, and a vector or transformed cell comprising the same.

[0013] Another aspect of the present invention relates to a method for inhibiting neutralization of a heterologous agent by neutralizing antibodies upon administration of the heterologous agent to a subject, the method 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.

[0014] A further aspect of the 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.

[0015] A further aspect of the invention relates to a method of treating a disorder in a subject in need thereof, where the disorder is treatable by expressing a polypeptide or functional nucleic acid in the subject, comprising 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.

[0016] Another aspect of the invention relates to a method of editing a gene in a subject, the method 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.

[0017] A further aspect of the present invention relates to a method of 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.

[0018] 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. Another aspect of the present invention relates to a method for treating and / or preventing acute transplant rejection of a solid organ associated with recognition of the transplant by recipient 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 pre- and / or post-transplant, thereby treating the acute transplant rejection.

[0019] 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 contacting the compound with a modified Mycoplasma protein M or a functional fragment thereof of the present invention attached to a solid support, and then eluting the compound from the modified Mycoplasma protein M or a functional fragment thereof.

[0020] A further aspect of the present invention relates to a method of performing an immunoassay, the method comprising using a modified Mycoplasmal 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.

[0021] A further aspect of the present invention relates to a kit comprising the modified Mycoplasmal protein M of the present invention or a functional fragment thereof.

[0022] These and other aspects of the invention are set forth in greater detail in the following description of the invention. [Brief description of the drawings]

[0023] [Figure 1]We show that human IVIG contains AAV neutralizing antibodies that inhibit AAV transduction in a dose-dependent manner. AAV2 neutralization plots were generated using two-fold dilutions of human IVIG. Starting at 50 μg, IVIG was serially diluted to less than 1 μg, and each dilution was incubated with 2×108 viral genomes of AAV2-luciferase for 1 h at 4°C before seeding into each well. The luminescent reporter signal generated by AAV2 transgene expression is a functional readout of AAV transduction and is proportional to the amount of AAV that was available to transduce cultured HEK-293 cells. In this experiment, 1×105 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 h after 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 at 12.5 μg IVIG compared to a no IVIG control with an equivalent volume of phosphate buffered saline (PBS). Furthermore, 25 μg IVIG neutralized 96% (± 0.2%) of AAV2 and 50 μg IVIG neutralized 99% (± 0.17%). [Diagram 2]We show that protein M protects AAV from neutralizing antibodies present in human IVIG. Using 12.5 μg IVIG, which was established in the previous figure to neutralize approximately 70% of AAV2 at a dose of 2x108 vg, escape from antibody-mediated neutralization was investigated using a 2-fold dilution ratio of protein M. In this case, we started the experiment with a molar ratio of 8 molecules of protein M to 1 molecule of IgG, which is a 4-fold excess of protein M, considering that one molecule of IgG has two protein M binding sites that should be occupied for complete antibody inactivation. In each well, IVIG was first incubated with the individual protein M dilutions for 1 h at 4 °C, followed by the addition of AAV2-luciferase (2x108 vg) and another hour of incubation at 4 °C. HEK-293 cells (1x105) were then added to the wells and, after 48 h of incubation, the luciferase reporter signal was measured. Wells containing IVIG but no protein M showed approximately a 68% (+ / - 3.3%) decrease in AAV signal compared to no-IVIG control wells containing phosphate-buffered saline (PBS). However, protein M dose-dependently blocked AAV neutralization, completely preventing neutralization (108%-193% of 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 no-IVIG control, respectively). Furthermore, a dose-dependent increase in AAV luciferase signal was observed at ratios of 8:1 and 4:1 over the no-IVIG control. All wells were performed in triplicate in serum-free medium (n=3 technical replicates per condition) in 48-well plates, and luciferase signal was measured 48 hours after transduction. [Diagram 3]We show 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 an upper limit for 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 IVIG neutralized the AAV2-luciferase signal by 80% (+ / - 2.6%) compared to PBS control wells without IVIG. As in the previous experiment, protein M with 12.5 μg 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 no IVIG control. At 10 times the amount of protein M required for NAb escape (20:1 molar ratio, 75 μg protein M), a slight increase in luciferase activity was observed to 256% (+ / - 44%) relative 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 the 8:1 ratio. Furthermore, without IVIG, when 75 μg or 33 μg protein M was incubated alone 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 1×105 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 4]The results show that protein M enhancement of AAV transduction is dose-dependent. Starting at 33 μg, two-fold dilution ratios of protein M show that incubation of protein M with AAV2-luciferase results in an increase in luciferase signal compared to the PBS+AAV control. The enhancement of luciferase signal disappears at concentrations of protein M below 2 μg for 2×10 8 viral genomes, i.e., at a molar ratio of protein M of 40,000 molecules per genome-containing AAV2-luciferase particle. The enhancement begins to saturate at approximately 33 μg protein M for 2×10 8 vector particles, or approximately 700,000 molecules of protein M for 1 genome-containing AAV2-luciferase particle. Protein M dilutions were incubated with AAV for 1 hour at 4° C. before transduction. Each well was seeded with 1×10 5 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). [Diagram 5]We show that protein M-based enhancement of AAV transduction depends on a direct interaction of protein M with the AAV capsid. Three different conditions were tested with AAV2-luciferase: pre-incubation of protein M with AAV 1 hour before transduction (-1 hour), addition of protein M to AAV at a time point close to transduction (0 hours), or addition of protein M to the wells 18 hours after transduction with AAV (18 hours). Two negative control conditions without protein M were used: AAV alone was incubated for 1 hour at 4°C in cell culture medium before cell seeding and transduction (representing the conditions in the pre-incubation and close to 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). 18 hours after transduction, an additional volume of PBS was added to the PBS control group to mimic the condition where an equivalent volume of protein M was added to the post-transduction group. No enhancement of AAV luciferase signal was seen 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 of AAV with protein M is required for the enhancement mechanism. All wells were seeded with 1x105 Huh7 cells and each well was transduced with 2x108 vg of AAV, with n=3 technical replicates per well condition in a 48-well plate format. [Figure 6]This shows that Protein M does not prevent AAV neutralization after NAb binds to the capsid. AAV2-luciferase was first incubated with different dilutions of IVIG for 1 h at 4° C., then Protein M was added and incubated for another 1 h at 4° C. The double negative control group received AAV only, while the positive control received 33 μg of Protein M incubated with AAV for 1 h at 4° C. before transduction. Three different dilutions of IVIG were used, including 200 μg, 50 μg, or 12.5 μg. Compared to the Protein M negative control group containing only AAV incubated with dilutions of IVIG, 33 μg of Protein M does not allow post-neutralization enhancement or escape from neutralizing antibodies when 99% of AAV is already neutralized by 50 μg or 200 μg of IVIG. However, when 12.5 μg IVIG was incubated with AAV, about 30% of the vector remained unneutralized (see Figures 1, 2, and 3), and 33 μg Protein M was able to enhance the transduction of this fraction after neutralization compared to Protein M negative control wells with AAV and 12.5 μg IVIG. Unlike the previous figures, luciferase measurements were performed only 24 hours after transduction, which could explain why the luciferase signal by the group with 12.5 μg IVIG + AAV was less than 30% of the double negative control. Each well was seeded with 1 × 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 7]We show that pre-incubation of AAV with protein M protects the vector from subsequent neutralization by IVIG. For this neutralization assay, the amount of protein M was kept the same (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 h at 4 °C, followed by addition of IVIG and incubation for 1 h at 4 °C before transducing cell cultures. Positive control groups contained AAV + 33 μg, 8.25 μg, or 1 μg of protein M, and 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 × 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). Luciferase measurements were performed 24 hours after transduction. [Figure 8]We show that pre-incubation of AAV with protein M protects the vector from neutralization by IVIG in the presence of excess background serum immunoglobulin. For this experiment, we used 25 μg of IVIG, which was previously shown to neutralize approximately 95% of AAV2-luciferase at a dose of 2 × 108 viral genomes. 25 μg of human IVIG was added to cell culture wells containing 10% fetal bovine serum (FBS) with an estimated bovine IgG concentration of 350 μg measured by an 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 h at 4 °C before adding 10% FBS to the no-IVIG control wells or to the wells containing 25 μg of IVIG, respectively. In negative controls, 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 IVIG. Results show that preincubation with protein M protected AAV2-luciferase from neutralization by IVIG even at a ratio of 1 molecule of protein M to 1 molecule of bovine IgG, but this amount of protein M (108 μg) was still a 12-fold higher ratio than human IVIG (25 μg). Each well was seeded with 1×105 HEK-293 cells 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 show that protein M allows AAV8 to escape in vitro from neutralizing antibodies found in pooled polyclonal sera collected from AAV8-immunized mice. For this experiment, 10 μl of pooled polyclonal sera from AAV8-immunized mice were serially diluted in PBS to produce wells containing 1 μl and 0.1 μl of serum, first diluted 10-fold. The 0.1 μl serum wells were then further diluted at 2-fold dilution ratios. AAV8-luciferase vectors (2×108 viral genomes per well) were incubated with dilutions of neutralizing sera at 4° C. for 1 hour before transduction. Protein M was added to the same 10-fold dilution ratio of AAV8-neutralized mouse sera, with an approximate ratio of 2 molecules of protein M to 1 molecule of immunoglobulin. Starting with 6.5 μg of Protein M for an estimated 10 μg of immunoglobulin in 1 μl of neutralizing mouse serum, each Protein M and serum sample was diluted against each other separately and then combined for 1 hour incubation at 4° C. All samples were then incubated with AAV8-luciferase for 1 hour at 4° C. before being added to cells. Data from the resulting neutralization experiments were normalized to no serum control wells containing only AAV8, and the luciferase signal was then fitted to a double exponential decay function to extrapolate a curve between the data points. Using the modeled curve, it was found that 50% neutralization of AAV8 by the polyclonal serum occurred in a volume of 0.0039 μl for 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 in a serum volume of 0.2744 μl, resulting in an estimated effective titer of 1:36. The results show that protein M is capable of protecting AAV across a nearly 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), with n=3 technical replicates per well condition, and luciferase was measured 48 hours after transduction. [Figure 10]We show that in vivo administration of protein M to mice with passive immunity to AAV8 results in neutralizing antibody escape. In this experiment, mice were given various volumes of polyclonal AAV8 serum (from previous figures, titer 1:2,564) by passive transfer via IV infusion and then, within 15-20 min, 2 x 1010 viral genomes of AAV8-luciferase were administered IV. This established a neutralization curve based on the serum volume delivered to each mouse. It was found that greater than 50% neutralization of the AAV8-luciferase signal occurred with 1 µl to 0.001 µl of transferred serum compared to a group of naive mice with no serum passively transferred. However, when an estimated 2:1 ratio of protein M (6.3 mg) was delivered to mice after passive transfer and before AAV administration, then complete escape from neutralizing antibodies was achieved with 0.3 μl transferred serum volume, and 1 μl showed protein M-mediated escape from neutralizing antibodies of 1,000-fold change in NAb concentration. Furthermore, escape from neutralization was dose-dependent for the 0.3 μl passively transferred serum group, indicating that NAb escape was reduced by an estimated molar ratio 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 per group, and luciferase signal from 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]It shows that Protein M / antibody complexes are stable after in vitro formation. In this experiment, 1 μl of polyclonal serum (titer 1:2,564) containing approximately 10 μg immunoglobulin was used to incubate with Protein M (2:1 molar ratio, 6.6 μg) for various durations before the addition of AAV8-luciferase. The negative control group contained neutralizing serum + medium, and the positive control group contained medium + PBS or Protein M + medium. Incubation intervals of 72 h, 48 h, 24 h, 16 h, 4 h, 2 h, and 1 h were applied to all groups. All incubation durations showed Protein M-mediated protection of AAV8 from neutralization compared to the negative control of medium + PBS. In a 96-well plate format, AAV8-luciferase was added to wells at the time of seeding (0 h) of 5 x 104 HEK-293 cells and transduced at an MOI of 4,000 (n = 3 technical replicates per well condition). Luciferase measurements were performed 48 h after transduction. [Figure 13] This shows that the efficacy of neutralizing antibody escape by Protein M in vivo is unstable. The same experiments as in Figures 10 and 11 were performed, but this time AAV8 was added 5 min after Protein M administration or 3 h after Protein M administration to test the durability of NAb escape. The AAV8-luciferase signal was about 1 / 3 of the no serum control group after waiting 3 h before administering AAV8, but when only 5 min was waited before administering AAV8 after Protein M, the luciferase signal was almost equivalent to the no serum control group. An estimated 2:1 ratio of Protein M (6.3 mg) was delivered to mice after passive transfer but before AAV administration, n=5 mice per group condition, except for the 3 h interval group, which contained n=3 mice. Luciferase signal from liver was measured 24 h after AAV administration. [Figure 14]We show that truncated protein M from M. genitalium (MG WT) is unstable at body temperature. Melting temperatures (Tm) were determined using nano-differential 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 showed that MG WT was stable at 20°C for at least 2 hours with no visible precipitates formed. However, at 37°C, MG WT began to unfold after 15 minutes with visible precipitates forming, indicating that the protein is unstable near standard human body temperature. A temperature gradient from 0 to 100°C showed immediate unfolding of MG WT and a melting temperature (Tm) of approximately 41.2°C. Unfolding was accompanied by aggregation with visible precipitates. [Figure 15A-15B] Melting temperatures are shown for truncated protein M from M. genitalium (MG WT) and M. pneumoniae (MP WT), as well as analogs of protein M that have at least one degree improvement in melting temperature over MG WT (A) or that maintain the melting temperature of MG WT (B). Melting temperatures were determined by differential scanning fluorography (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 a range of thermal stabilities, with multiple mutations producing additive effects to increase melting temperatures. [Figure 16] Example data is 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 here 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 soluble fraction assessment 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 soluble fraction was run on an SDS-PAGE gel after pelleting precipitated protein by centrifuging the samples at 15,000 x G for 10 min. Proteins were heated for 0, 1, 4, 24, 48, 72, or 96 hours prior to assessment. Results show that MG WT (A) begins to precipitate out of solution after 1-4 hours of heating. MG27 (B) and MG29 (A) remain soluble over 72 hours, while MG31 (B) and MG40 (C) remain soluble over 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 vector during in vitro neutralization assays: comparison of 4°C vs. 37°C heat challenge for 1 h (A and B) or 24 h (C) incubation. Protein M vs. IVIG at a 4:1 ratio. Relative light produced by luciferase activity was measured from cell cultures 24 h after transduction with 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 h showed nearly complete neutralization of AAV. Protein M analog (16 μg) incubated at 4°C prevented neutralization of AAV by IVIG when incubated together for 1 h prior to AAV. This was compared to Protein M analogs incubated at 4°C without incubation with IVIG (not performed for MG8 and MG24). When the analogs were heat challenged at 37°C prior to incubation with IVIG, MG WT and some analogs with lower melting temperatures did not protect AAV from neutralization, while other analogs with higher Tm maintained the ability to protect AAV from neutralization. Most mutant MG analogs maintained the ability to block neutralizing antibodies after 1 hour of 37°C challenge. However, MG27, MG29, and MG46 prevented AAV neutralization after 24 hours of 37°C challenge. [Figure 19]We show that MG WT blocks NAb upon AAV rechallenge one month after primary AAV administration. Wild-type C57BL6 female mice were immunized via intraperitoneal administration of AAV8-GFP (1E9vg) and serum was collected one month later to assess AAV neutralizing antibody titers in vitro. Mice were then intramuscularly administered AAV8-luciferase reporter vector (1E9vg / leg) where AAV was formulated as a simple mix with MG WT for the right leg of the mouse (33 μg / leg) or with phosphate-buffered saline as a vehicle control for the left leg of the mouse. Luminescence imaging of leg muscles was performed two weeks after AAV8-luciferase administration. Three mice with AAV neutralizing antibody titers 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 leg produced an 80% lower mean luciferase signal than in the MG WT-formulated leg. This result indicates 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 show that MG29 blocks NAb upon AAV rechallenge one month after primary AAV administration. Wild-type C57BL6 female mice were immunized via intraperitoneal administration of AAV8-GFP (5E8vg) and serum was collected one month later to evaluate AAV neutralizing antibody titers in vitro. Mice were then intramuscularly administered AAV8-luciferase reporter vector (2E9vg / leg), where AAV was formulated as a simple mix with engineered analog MG29 for the right leg of the mouse (500 μg / leg) or with phosphate-buffered saline as a vehicle control for the left leg of the mouse. 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 leg resulted in a mean luciferase signal that was at least 98% lower than in the MG WT-formulated leg. This result indicates that AAV can be successfully re-administered using engineered Protein M analogs after the production of neutralizing antibodies induced by a previous AAV administration. [Figure 21] Figure 1 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 the BLI probe 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]Success rates of mutant stabilization are shown, representing the frequency with which mutations predicted by Rosetta for MG WT increased (Tm+1°C), decreased (Tm-1°C) stability, or had no effect. "Combination mutations" represent constructs constructed by merging mutation constructs that individually stabilized MG WT. [Figure 24] Conservation of the MP WT sequence is shown. Homology models of MP WT are shown in two orientations and colored by conservation 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. [Diagram 25] We show that codon optimization of the DNA sequence of protein M results in higher production yields. DNA plasmids encoding 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 original or optimized PM plasmids were cultured and grown in equivalent growth medium volumes (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. Crude lysates were centrifuged and supernatants were collected for protein separation on SDS-PAGE gels. Equal volumes from each lysate were run on SDS-PAGE gels and transferred onto Western blots and 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 MG WT protein was measured using a luminol chemiluminescence assay. The obtained MG WT yield was calculated based on the Western blot band intensity normalized to the weight of the bacterial pellet. The optimized PM plasmid showed a nearly 4-fold increase in MG WT protein yield when compared to the original PM plasmid. [Figure 26]Improved pH stability of the mutants is shown. Melting temperatures (Tm) were determined using NanoDSF at different pH conditions. Proteins were purified via SEC in PBS and buffer exchanged into glycine (pH 2.5 and 3.5), acetate (pH 4.5 and 5.5), and phosphate (pH 6.5 and 7.5) buffers. [Figures 27A-27D] 1 shows a sequence alignment of wild-type M. genitalium protein M amino acids 74 to 479 (SEQ ID NO: 3) and modified proteins 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 to 479 (SEQ ID NO: 3) and the equivalent fragment of M. pneumoniae protein M (SEQ ID NO: 23). [Figure 29]Figure 29 shows in vivo antibody neutralization 7 days after administration. Sera were collected and pooled from mice serially immunized with AAV8. In vitro neutralization assays determined that the anti-AAV8 neutralization titer of the pooled sera was approximately 1:10,000. Different amounts of anti-AAV8 serum (determined by serum volume) were administered via IV injection to groups of AAV naive mice in passive transfer experiments, followed 20–25 minutes later by IV injection of 2e10 viral genomes (vg) of the AAV8-luciferase reporter vector. Mice in some groups were administered protein M via IV injection 5 minutes prior to AAV administration. Noninvasive in vivo imaging of luciferase activity was performed 7 days later to quantify luminescence as a proxy for gene expression from the AAV8 vector, with a reduction in luminescence signal indicating AAV8 luciferase neutralization by serum. Mice given 0.0003ul of serum failed to neutralize AAV8, resulting in nearly 100% luminescent signal compared to serum-free naïve mice injected with only 2e10vg of AAV8-luciferase. Groups of mice given increasing amounts of neutralizing serum progressively neutralized AAV8-luciferase, with approximately half of the AAV neutralized with 0.001ul to 0.003ul of serum and over 99% of the AAV neutralized with 0.3ul or more of serum. Groups of mice given 3ul of serum, followed by MG88 or MG118, then 2e10vg of AAV8-luciferase resulted in gene expression indicative of escape from neutralization due to suppression of neutralizing antibodies by the protein M fusion proteins MG29 (6.3mg), MG29* (6.3mg), and MGWT* (6.3mg), MG118 (9mg), and MG88 (6.3mg). [Diagram 30]In vitro neutralization assays are shown in which high titer anti-AAV8 serum from mice was titrated at 2-fold dilutions and added to wells as indicated on the x-axis. Different Protein M analogs (MG29, MG66, MG71, and MG64) were then added to the wells at the molecular ratios indicated in the figure legend and incubated for 1 h. Saline was added to serum-only wells (black circles). AAV8-luciferase (2x1010 viral genomes) was then added to each well and incubated for 1 h before adding 5x104 Huh7 cells in serum-free medium. Cells were cultured for 48 h and the luciferase reporter signal, a surrogate for neutralization, was read and quantified. These data indicate that these analogs have neutralizing antibody blocking function, thereby shifting the neutralization curve, with the MG64 Fc-Protein M fusion dimer (Fc-PM) shifting the curve more than the Protein M monomer analog. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] 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 implemented 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, and do not depart from the invention. Thus, the following specification is intended to describe some particular embodiments of the invention, and is not intended to exhaustively specify all permutations, combinations, and variations thereof.

[0025] Unless the context dictates otherwise, it is expressly intended that the various features of the invention described herein may be used in any combination. 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. For illustrative purposes, if the present specification states that a complex comprises components A, B, and C, it is specifically intended that any or any combination of A, B, or C may be omitted and disclaimed, either alone or in any combination.

[0026] 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 terms used in the description of the present invention in this specification are for the purpose of describing particular embodiments only and are not intended to be limiting of the present invention.

[0027] Nucleotide sequences are presented herein as single strands only, in a 5' to 3' orientation, from left to right, unless expressly indicated otherwise. Nucleotides and amino acids are represented herein in the form 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 C.FR § 1.822 and established usage.

[0028] Unless otherwise indicated, standard methods known to those of skill in the art may 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 Edition (Cold Spring Harbor, NY, 1989); FMAUSUBEL et al. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York).

[0029] All publications, patent applications, patents, nucleotide sequences, amino acid sequences, and other references mentioned herein are incorporated by reference in their entirety.

[0030] 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.

[0031] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted in the alternative ("or").

[0032] Furthermore, the present invention also relates to a method for producing a pharmaceutical composition comprising the steps of: It is also contemplated that any feature or combination of features described herein may be excluded or omitted.

[0033] Furthermore, as used herein, the term "about" when referring to a measurable value, such as an amount, dosage, time, temperature, and the like, 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.

[0034] The transitional phrase "consisting essentially of" as used herein should be construed 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, the term "consisting essentially of" as used herein should not be construed as the equivalent of "comprising."

[0035] The term "consisting essentially of" (and grammatical variations) as applied to a polynucleotide or polypeptide sequence of the invention means a polynucleotide or polypeptide consisting of both a recited sequence (e.g., SEQ ID NO:) and a total of 10 or less (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 less additional nucleotides or amino acids includes the total number of additional nucleotides or amino acids taken together. The term "substantially altered" as 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" as 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.

[0036] The term "parvovirus" as used herein encompasses the Parvoviridae family, including 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 of skill in the art. See, e.g., FIELDS et al., VIROLOGY, Vol. 2, Chapter 69 (4th ed., Lippincott-Raven Publishers).

[0037] The Dependovirus genus includes adeno-associated viruses (AAV), 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.

[0038] 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). Many additional AAV serotypes and clades have been identified (see, e.g., Gao et al., (2004) J. Virol. 78:6381-6388 and Table 1), which are also encompassed by the term "AAV."

[0039] Parvovirus particles and genomes of the present invention may be derived from, but are not limited to, AAV. The genomic 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 may be found in the literature or in 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, J01901, J022 See, e.g., US Pat. Nos. 75, 801, 752, 753, 754, 755, 756, 757, 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. al.,(2002)Proc.Nat.Acad.Sci.USA99:11854, 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 al. See also, Schmidt et al., (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 Publication Nos. 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 the AAV1, AAV2, and AAV3 ITR sequences is provided by Xiao, X., (1996), "Characterization of Adeno-associated virus (AAV) DNA replication and integration", PhD dissertation, University of Pittsburgh, Pittsburgh, PA (incorporated herein in its entirety).

[0040] 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.

[0041] The term "tropism" as used herein refers to the preferential, but not necessarily exclusive, entry of a vector (e.g., a viral vector) into a particular cell or tissue type 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 (which may be, for example, transcription, translation) of sequences carried by the vector contents (e.g., the viral genome) in the cell, e.g., for recombinant viruses, expression of heterologous nucleotide sequences. Those skilled in the art understand that transcription of heterologous nucleic acid sequences from a viral genome cannot be initiated in the absence of trans-acting factors, e.g., for inducible promoters or otherwise regulated nucleic acid sequences. In the case of the rAAV genome, gene expression from the viral genome may be from a stably integrated provirus and / or from non-integrated episomes and any other form that viral nucleic acid may take in a cell.

[0042] The term "tropism profile" refers to the transduction pattern of one or more target cells, tissues and / or organs. Representative examples of chimeric AAV capsids have a tropism profile characterized by efficient transduction of cells of the central nervous system (CNS) with little transduction of peripheral organs (see, e.g., U.S. Pat. No. 9,636,370 (McCown et al.), and U.S. Patent Publication No. 2017 / 0360960 (Gray et al.)). Vectors (e.g., viral vectors, e.g., AAV capsids) that express specific tropism profiles can be referred to as "tropism" for their tropism profile, e.g., neurotropism, liver tropism, etc.

[0043] As used herein, "transduction" of a cell with a viral vector (e.g., an AAV vector) refers to entry of the vector into the cell and 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. [Table 1] JPEG2024505948000003.jpg48166

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

[0045] Similarly, a virus "does not efficiently transduce" or "does not have efficient tropism" for a target tissue, or similar terms, can be determined by reference to a suitable control. 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 a tissue (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 a desired target tissue (e.g., CNS cells).

[0046] The terms "5' portion" and "3' portion" are relative terms 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 the 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 the 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] The term "polypeptide" as used herein, unless otherwise indicated, includes both peptides and proteins.

[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- or double-stranded DNA sequence.

[0049] The term "regulatory element" refers to a genetic element that controls some aspect 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 may be referred to as a "regulatory region."

[0050] The term fragment, when applied to a polypeptide, refers to an amino acid sequence that is reduced in length compared to a reference polypeptide or amino acid sequence and will be understood to comprise, consist essentially of, and / or consist of an amino acid sequence of identical contiguous amino acids as the reference polypeptide or amino acid sequence. In some embodiments, such a fragment may 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 contiguous 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 (e.g., antibody binding) normally associated with the polypeptide. A "functional fragment" substantially maintains all of the activities of the unmodified polypeptide. By "substantially maintain" biological activity, it is meant 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] The term "operably linked" as used herein 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) for initiating transcription of the polypeptide. The term "coding region" may be used interchangeably with open reading frame.

[0054] The term "codon optimization" as used herein 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 nucleobase sequences of the underlying 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 synonymous codons that occur more frequently 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 expression level of the protein 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] The term "sequence identity" as used herein 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 may 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), the sequence identity alignment algorithm of Needleman & Wunsch, J Mol. Biol. 48:443 (1970), the search for similarity method of Pearson & Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA, Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), the Best Fit sequence program described by Devereux et al., Nucl. Acid Res. 12:387 (1984), preferably using 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), which is similar to the method described in 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, obtained from Altschul et al., Meth. Enzymol., 266:460 (1996); 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, 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, the values ​​can also be adjusted to increase sensitivity.

[0058] An additional 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 of the "longer" sequence in the aligned region, the "longer" sequence being the one with the most actual residues in 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 sequences being aligned. Furthermore, with respect to sequences that contain more or fewer nucleotides than the polynucleotides specifically disclosed herein, it is understood that 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 is, in one embodiment, determined using the number of nucleotides in the shorter sequence. In calculating the percentage of identity, no relative weight is assigned to the various occurrences of sequence variations, such as insertions, deletions, substitutions, 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 sequence 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 from at least a portion of other components of an organism or virus in which it naturally originates, e.g., a structural component of a cell or virus or other polypeptides or nucleic acids that are typically found in association with the nucleic acid or nucleotide sequence.

[0064] Similarly, an "isolated" polypeptide means a polypeptide that is separated or substantially free from 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 typically found in association with a structural component or polypeptide of a cell or virus.

[0065] As used herein, the term "modified" when applied to a polynucleotide or polypeptide sequence, refers to a sequence that differs from the wild-type sequence by 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 their grammatical equivalents) mean to delay or inhibit the onset of a disease. The term is not meant to require complete elimination of the disease, but rather encompasses any type of prophylactic treatment to reduce the incidence of symptoms or to delay the onset of symptoms.

[0069] As used herein, a "therapeutically effective" amount is an amount sufficient to provide some improvement or benefit to a subject. In other words, a "therapeutically effective" amount is an amount that provides some relief, alleviation, reduction or stabilization of at least one clinical symptom in a subject. Those skilled in the art understand that the therapeutic effect does not have to 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 condition in a subject and / or reduce the severity and / or delay the onset of a disease, disorder, and / or clinical condition in a subject compared to that which would occur in the absence of the method of the 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 non-translated RNA.

[0072] "Vector" refers to a compound used as a vehicle to carry foreign genetic material to 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. A recombinant vector typically contains 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. Expression vectors (expression constructs or expression cassettes) are for the expression of exogenous genes in target cells and generally have a promoter sequence that drives the expression of the exogenous gene / ORF. The insertion of a vector into a target cell is called transformation or transfection for bacterial and eukaryotic cells, while the 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 to 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 a viral particle that, in certain embodiments, functions as a nucleic acid delivery vehicle and contains viral nucleic acid (i.e., vector genome) packaged within the virion. A viral vector of the present invention comprises a chimeric AAV capsid of the present invention and can package an AAV or rAAV genome or any other nucleic acid (including viral nucleic acid). Alternatively, in some contexts, the terms "vector," "viral vector," "delivery vector" (and similar terms) may be used to refer to a virion-free vector genome (e.g., vDNA) and / or a viral capsid that acts as a vehicle to deliver a molecule 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 No. 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) that contains at least one inverted terminal sequence (e.g., one, two, or three inverted terminal sequences) 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 that contain 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). A rAAV vector may 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 it. The TRs may be identical or different to 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 terminal repeat or synthetic 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), human parvovirus B-19), or the SV40 hairpin acting as an SV40 origin of replication, may be used as a TR, which may be further modified by truncation, substitution, deletion, insertion, and / or addition. Additionally, the TR may be partially or completely synthetic, such as the "double-D sequence" described in U.S. Patent No. 5,478,745 to 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. The hairpin structure is believed to adopt 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 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 the native ITR sequence (e.g., the native AAV ITR sequence 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. A "rAAV particle" or "rAAV virion" comprises a rAAV vector genome packaged within an AAV capsid.

[0080] The viral vectors of the present invention may further be "targeted" viral vectors (e.g., having directed tropism) and / or "hybrid" parvoviruses (i.e., in which the viral ITRs and viral capsid are derived from different parvoviruses) as described in International Patent Publication No. WO 00 / 28004 and Chao et al., (2000) Mol. Therapy 2:619.

[0081] In addition, the viral capsid or genomic elements can 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 shown in Table 2. [Table 2]

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

[0085] Additionally, the unnatural amino acid can be a "non-natural" amino acid as described by Wang et al., (2006) Annu. Rev. Biophys. Biomol. Struct. 35:225-49. These unnatural amino acids can be advantageously used to chemically link a molecule of interest to an AAV capsid protein.

[0086] Conservative amino acid substitutions are known in the art. In certain embodiments, conservative amino acid substitutions include substitutions in 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.

[0087] The term "template" or "substrate" is used herein to refer to a polynucleotide sequence that can be replicated to produce the viral DNA of a parvovirus. For purposes of vector production, the template is typically embedded within a larger nucleotide sequence or construct, including, but not limited to, a plasmid, a naked DNA vector, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), or a viral vector (e.g., adenovirus, herpes virus, Epstein-Barr virus, AAV, baculovirus, retroviral vectors, etc.). Alternatively, the template can be stably integrated into the chromosome of the packaging cell.

[0088] 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 viral particles. Parvovirus and AAV replication genes and proteins are described, for example, in FIELDS et al., VIROLOGY, Vol. 2, Chapters 69 and 70 (4th ed., Lippincott-Raven Publishers).

[0089] A "Rep coding sequence" need not code for all of the parvovirus or AAV Rep proteins. For example, with respect to AAV, the Rep coding sequence need not code for all four AAV Rep proteins (Rep78, Rep68, Rep52 and Rep40), and indeed, AAV5 is believed to express only the spliced ​​Rep68 and Rep40 proteins. In representative embodiments, the Rep coding sequence codes for at least the replication proteins required for viral genome replication and packaging into new virions. The Rep coding sequence generally codes for 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 codes for the AAV Rep78 protein, as well as the AAV Rep52 and / or Rep40 proteins. In other embodiments, the Rep coding sequence codes for 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.

[0090] The term "large Rep protein" as used herein refers to Rep68 and / or Rep78. The large Rep proteins of the claimed invention may be either wild-type or synthetic. Wild-type large Rep proteins may 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 may be altered by insertions, deletions, truncations, and / or missense mutations.

[0091] Those skilled in the art will further appreciate that the replication proteins do not have to be encoded by the same polynucleotide. For example, for MVM, the NS-1 and NS-2 proteins (splice variants) can be expressed independently of each other. Similarly, for AAV, the p19 promoter can be inactivated, the large Rep protein can be expressed from one polynucleotide, and the small Rep protein can be expressed from a different polynucleotide. However, it is typically more convenient to express the 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 and small Rep proteins from separate expression cassettes. In other instances, it may be desirable to express the large and small Rep proteins 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 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).

[0092] As used herein, a parvovirus or AAV "cap coding sequence" encodes 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 less 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.

[0093] The capsid structure of autonomous parvoviruses and AAV is described in further detail in BERNARD N. FIELDS et al., VIROLOGY, Vol. 2, Chapters 69 and 70 (4th ed., Lippincott-Raven Publishers).

[0094] 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.

[0095] 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 upon administration of the heterologous agent to a subject, the method comprising administering to a subject an effective amount of Mycoplasma protein M, or a functional fragment or derivative thereof, or a fusion protein comprising an effective amount of Mycoplasma protein M, or a functional fragment or derivative thereof, thereby inhibiting neutralization of the heterologous agent.

[0096] Another aspect of the invention relates to a method of 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, or a fusion protein comprising Mycoplasma protein M or a functional fragment or derivative thereof, thereby expressing the polypeptide or functional nucleic acid in the subject.

[0097] A further aspect of the invention relates to a method of editing a gene in a subject, the method 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, or a fusion protein comprising 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.

[0098] The term "heterologous agent" as used herein 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 factors 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 the subject. A heterologous agent may have never been administered to a subject. A heterologous agent may have been previously administered to a subject.

[0099] The term "neutralizing antibody" as used herein 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.

[0100] In some embodiments, the heterologous agent may be a nucleic acid delivery vector, for example, a viral vector or a non-viral vector. In some embodiments, the viral vector is an oncolytic vector. In some embodiments, the viral vector is an adeno-associated virus, a retrovirus, a lentivirus, a poxvirus, an alphavirus, a baculovirus, a vaccinia virus, a herpes virus, an Epstein-Barr virus, a poliovirus, or an adenovirus vector. In some embodiments, the non-viral vector is a plasmid, a liposome, a charged lipid, a nucleic acid-protein complex, or a biopolymer.

[0101] In some embodiments, the heterologous agent is a gene editing complex, such as a CRISPR complex.

[0102] 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.

[0103] An effective amount of protein M or a functional fragment or derivative thereof, or a fusion protein comprising an effective amount of mycoplasma protein M or a functional fragment or derivative thereof, is an amount that at least partially blocks the inhibition of a heterologous agent by a neutralizing antibody and / or overcomes the ability of an autologous neutralizing antibody to bind to a heterologous agent. In some embodiments, an effective amount of protein M or a functional fragment or derivative thereof 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, the effective amount of protein M or a functional fragment or derivative thereof, or a fusion protein comprising an effective amount of mycoplasma 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, such as 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 other embodiments, the ratio is about 8:1 to about 50:1. In some embodiments, the ratio is from about 0.5:1 to about 6:1, from about 0.5:1 to about 4:1, from about 0.5:1 to about 2.5:1, from about 0.5:1 to about 2:1, from about 1:1 to about 8:1, from about 1.5:1 to about 8:1, or from about 2:1 to about 8:1. In one embodiment, the ratio is from about 1:1 to about 3:1, e.g., about 2:1.

[0104] In some embodiments, administration comprises about 1e9vg / kg to about 1e14vg / kg, about 1e10vg / kg to about 1e13vg / kg, or about 1e11vg / kg to about 1e12vg / kg of AAV, about 1 μl to about 6 L of serum, and about 1mg / kg to about 1000mg / kg, about 10mg / kg to about 900mg / kg, about 25mg / kg to about 800mg / kg, about 50mg / kg to about 600mg / kg, about 100mg / kg to about 500mg / kg, or about 200mg / kg to about 400mg / kg of Protein M.

[0105] Total immunoglobulin may be total serum immunoglobulin (e.g. for systemic administration of Protein M). Total immunoglobulin may be the total level in a localized fluid or tissue (e.g. for specific delivery to eye, ear, lung, brain, muscle, joint, etc.). Total immunoglobulin may be measured by any technique known in the art, for example, by performing an ELISA on the serum using an antibody that binds to the Fc region of the immunoglobulin, or by using protein A or G that binds to the immunoglobulin. Furthermore, for mice, their serum is known to contain 5mg / ml to 10mg / ml of immunoglobulin. The normal range for serum immunoglobulin in humans is 8 to 10mg / ml. For in vivo estimations, a high end of 10mg / ml can be used to calculate the ratio. Local immunoglobulin content can be estimated based on tissue weight (40mL serum / kg body weight), or the concentration of Ig in a particular body fluid, and the volume of the fluid in that organ if less than blood serum (e.g. eye, cerebrospinal fluid).

[0106] Protein M may 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, or a fusion protein comprising an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, is administered to a subject prior to administration of a heterologous agent, for example, 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, 24, 48, or 72 hours prior to administration of a heterologous agent. In some embodiments, protein M or a functional fragment or derivative thereof, or a fusion protein comprising an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, is administered to a subject simultaneously with administration of a heterologous agent. As used herein, the term "concurrently" means close enough in time to produce a combined effect (i.e., "concurrently" can mean "simultaneously" or two or more events occurring one after the other within a short period of time).

[0107] In some embodiments, the heterologous agent is combined with protein M or a functional fragment or derivative thereof, or a fusion protein comprising an effective amount of Mycoplasma 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 can be combined with protein M or a functional fragment or derivative thereof, or a fusion protein comprising an effective amount of Mycoplasma 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, or a fusion protein comprising an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, and the heterologous agent are administered in separate compositions.

[0108] In some embodiments, it may be necessary to administer the heterologous agent and / or protein M or a functional fragment or derivative thereof, or a fusion protein comprising an effective amount of mycoplasma protein M or a functional fragment or derivative thereof, to the subject more than once to provide a therapeutic or beneficial effect. Protein M or a functional fragment or derivative thereof, or a fusion protein comprising an effective amount of mycoplasma protein M or a functional fragment or derivative thereof, may be administered, for example, one, two, three, four or more times. Administration may be spaced apart by days, weeks, months, or years (e.g., from about 2 days to about 10 years or more). In some embodiments, administration reduces the amount of antibody cycling, with the onset of the reduction being about 1 hour to about 3 hours. In some embodiments, administration reduces the amount of antibody cycling, with the onset of the reduction being about 5 minutes to about 8 weeks, about 5 minutes to about 1 hour, about 1 hour to about 2 days, or about 2 days to about 8 weeks. In some embodiments, the protein M or a functional fragment or derivative thereof, or a fusion protein comprising an effective amount of the Mycoplasma protein M or a functional fragment or derivative thereof, is administered to the subject each time a heterologous agent is administered to the subject, for example, in a manner similar to that described above, for example, before or at the same time as the heterologous agent. The use of protein M with each administration of a heterologous agent may have NAb effects against the heterologous agent, which is often problematic when re-administered. In some embodiments, the same protein M or a functional fragment or derivative thereof, or a fusion protein comprising an effective amount of the Mycoplasma 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, or a fusion protein comprising an effective amount of the Mycoplasma protein M or a functional fragment or derivative thereof, for example, a different modified protein M, as further described below, is administered each time. Without being bound by theory, it is believed that the use of a different protein M derivative in each administration may limit the effect of inhibitory antibodies against protein M that may occur with 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, or a fusion protein comprising an effective amount of mycoplasmal protein M or a functional fragment or derivative thereof, will overcome any inhibitory antibodies against protein M and prevent antigen recognition. In some embodiments, administration overcomes autologous neutralizing antibodies.

[0109] The ability of protein M or a functional fragment or derivative thereof, or a fusion protein comprising an effective amount of mycoplasma protein M or a functional fragment or derivative thereof, to bind non-specifically to antibodies may be advantageously used in other methods in which it is beneficial to inhibit antibody binding to an antigen, for example when immunosuppression is desired or when an excess of antibodies is present.

[0110] A further aspect of the invention relates to a method of 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, or a fusion protein comprising an effective amount of Mycoplasma protein M, or a functional fragment or derivative thereof, thereby treating the autoimmune disease.

[0111] As used herein, the term "autoimmune disease" refers to any disorder associated with an autoimmune response. Examples include multiple sclerosis, Crohn's disease, ulcerative colitis, systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel syndrome, irritable bowel syndrome, uveitis, insulin-dependent diabetes mellitus, hemolytic anemia (e.g., warm autoimmune hemolytic anemia), rheumatic fever, Goodpasture's syndrome, Guillain-Barré syndrome, psoriasis, thyroiditis, Graves' disease, myasthenia gravis, glomerulonephritis, autoimmune hepatitis, immune thrombocytopenic purpura, acute inflammatory demyelinating purpura, and acute myelinating purpura. These include, but are not limited to, polyneuropathy, antibody-mediated rapidly progressive glomerulonephritis, chronic inflammatory demyelinating polyradiculoneuropathy, hyperviscosity syndrome, relapsing focal segmental glomerulosclerosis, neuromyelitis optica spectrum disorder, cryoglobulinemia, autoimmune encephalitis, autoimmune neuropathy, ANCA vasculitis, autoimmune neutropenia, anti-GBM disease, pemphigus vulgaris, Sjogren's syndrome, lupus nephritis, and immune cytopenias.

[0112] 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, or a fusion protein comprising an 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 "disorder associated with excess antibodies" refers to any disorder in which the cause or at least one symptom of the disorder is due to higher than average antibody levels in the blood or elsewhere in the body. Examples include, but are not limited to, multiple myeloma, monoclonal gammopathy of undetermined significance (MGUS), Waldenstrom's macroglobulinemia, cytokine release syndrome, or acute autoimmune attacks, such as sudden onset severe autoimmune vasculitis. The methods may also be useful for acute blocking of all antibodies to rapidly stop an autoimmune event, e.g., cytokine release syndrome or an acute autoimmune attack, e.g., sudden onset severe autoimmune vasculitis, or to prevent damage to transplanted tissues and / or organs caused by antibody-mediated immune complex formation.

[0113] For any of the methods of the present invention, protein M or a functional fragment or derivative thereof, or a fusion protein comprising an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, can be administered to a subject by any route of administration (e.g., locally or systemically) known to be effective.

[0114] The most suitable route depends on the subject to be treated and the disorder or condition to be treated. In some embodiments, protein M or a functional fragment or derivative thereof, or a fusion protein comprising an effective amount of mycoplasma 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., by aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, intravitreal, intracochlear, transdermal, intraendothelial, intrauterine (or intraembryo), 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 airway surfaces), and transdermal administration), intralymphatic, etc., and direct tissue or organ injection (e.g., liver, eye, skeletal muscle, cardiac muscle, diaphragm muscle, or brain).

[0115] Protein M or a functional fragment or derivative thereof, or a fusion protein comprising an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, can be delivered or targeted to any tissue or organ of a subject. In some embodiments, protein M or a functional fragment or derivative thereof, or a fusion protein comprising an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, is administered to, for example, skeletal muscle, smooth muscle, heart, diaphragm, airway epithelium, liver, kidney, spleen, pancreas, skin, lung, ear, and eye. In some embodiments, protein M or a functional fragment or derivative thereof, or a fusion protein comprising an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, is administered to a diseased tissue or organ, for example, a tumor.

[0116] In some embodiments, the heterologous agent and protein M or a functional fragment or derivative thereof, or a fusion protein comprising an effective amount of Mycoplasma 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, or a fusion protein comprising an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, are administered by different routes, for example, protein M or a functional fragment or derivative thereof, or a fusion protein comprising an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, is administered intravenously and the heterologous agent is administered locally to a target tissue or organ (e.g., a diseased tissue or organ).

[0117] Any of the above methods may further include 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 plasma exchange, administration of an antibody digestive enzyme such as IdeS or IdeZ, administration of an antibody that binds to the FcRn receptor, administration of an Fc fragment that binds to the FcRn receptor, 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, mycophenolic acid), or biologics (e.g., abatacept, adalimumab, anakinra, certolizumab, etanercein, erythropoietin ... Additional treatments include, but are not limited to, administration of anti-B-cell antibodies (e.g., ribavirin, golimumab, infliximab, ixekizumab, natalizumab, rituximab, secukinumab, tocilizumab, ustekinumab, vedolizumab, basiliximab, daclizumab), administration of antibodies that bind to CD19 on B cells or that bind to and deplete CD20, or other treatments designed to inhibit or destroy B cells (e.g., splenectomy, chemotherapy, immunotherapy, radiation therapy). Additional treatments may be administered before, during, and / or after administration of protein M or a functional fragment or derivative thereof, or a fusion protein comprising an effective amount of mycoplasma protein M or a functional fragment or derivative thereof.

[0118] The ability of protein M or a functional fragment or derivative thereof, or a fusion protein containing an effective amount of mycoplasma protein M or a functional fragment or derivative thereof, to bind non-specifically to antibodies can be advantageously 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 non-specifically to the variable region of antibodies. Thus, protein M can be used to isolate antibody fragments and antibody derivatives that do not contain the Fc region (e.g., single-chain variable fragments) and other molecules that incorporate antibody variable regions.

[0119] Thus, one aspect of the present invention is a method for isolating a compound comprising an antibody light chain variable region and / or heavy chain variable region from a sample, comprising contacting the compound with a modified Mycoplasma protein M or a functional fragment thereof of the present invention attached to a solid support, or a fusion protein comprising an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, and then eluting the compound from the modified Mycoplasma protein M or a functional fragment thereof or a fusion protein comprising an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof. In some embodiments, the compound comprising an antibody light chain variable region and / or heavy chain variable region is an antibody or an 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, etc. The modified protein M or a functional fragment thereof of the present invention, or a fusion protein comprising an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, is advantageous over wild-type protein M due to 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.

[0120] The method may be performed using techniques well known in the field of affinity purification. The solid support may 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.

[0121] The contacting step can be carried out by any suitable method, for example, by passing a sample containing the compound through a column containing the modified Mycoplasma protein M or a functional fragment thereof, or a fusion protein containing an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, or by incubating a composition containing the compound with the modified Mycoplasma protein M or a functional fragment thereof, or a fusion protein containing an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, in a well of a container or plate. The contacting step can be carried out for a time sufficient to allow the compound to bind to the modified Mycoplasma protein M or a functional fragment thereof, or a fusion protein containing an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof. After washing, centrifugation, or other form of separation that separates the compound bound to the modified Mycoplasma protein M or a functional fragment thereof, or a fusion protein containing an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof from other components in the sample, the compound is eluted from the modified Mycoplasma protein M or a functional fragment thereof, or a fusion protein containing an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof. Elution can be performed by any method known in the art, such as by changing ion concentration, temperature, etc. In one embodiment, elution can be performed by changing pH. The modified Mycoplasma protein M or functional fragments thereof of the present invention are advantageously stable over a wider range of pH than wild-type protein M. This allows the modified protein M to remain stable at low pH, which allows elution of the compound.

[0122] In some embodiments, the contacting step is carried out in a binding buffer (e.g., at neutral pH) and elution is carried out with a low pH buffer (e.g., 0.1 M glycine pH 2-3.5 or 0.1 M acetate pH 3.5-4.5) in a neutralizing buffer (e.g., a high ionic strength alkaline buffer such as 1 M phosphate or 1 M Tris at pH 8-9).

[0123] A further aspect of the invention relates to a fusion protein comprising a modified Mycoplasma protein M or a functional fragment thereof, or an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, attached to a solid support as described above. The modified Mycoplasma protein M or a functional fragment thereof, or a fusion protein comprising an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, can be attached to the solid support by any means known in the art, for example, covalently, for example, using a linker molecule.

[0124] The ability of protein M or a functional fragment or derivative thereof, or a fusion protein comprising an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, to bind advantageously and non-specifically to an antibody can be used in any immunoassay method involving binding an antibody, a fragment or derivative thereof, or a fusion protein comprising an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof.

[0125] Thus, one aspect of the present invention relates to a method of performing an immunoassay, the method comprising using a modified Mycoplasmal protein M of the present invention, or a functional fragment thereof, or a fusion protein comprising an effective amount of Mycoplasmal protein M, or a functional fragment or derivative thereof, to bind to a compound comprising an antibody light chain variable region and / or heavy chain variable region.

[0126] A fusion protein comprising a modified Mycoplasma protein M or a functional fragment thereof, or an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, can replace any general or specific antibody binding molecule, such as Protein A, Protein G, or a secondary antibody. A modified Mycoplasma protein M or a functional fragment thereof, or a fusion comprising an effective amount of Mycoplasma protein M or a functional fragment or derivative thereof, can be labeled for detection, for example, radioactively, chemiluminescently, or enzymatically, as is well known in the art.

[0127] Examples of immunoassays include, but are not limited to, radioimmunoassays (RIA), enzyme-linked immunosorbent assay (ELISA) assays, enzyme-linked immunoassays (EIA), 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, immunoprecipitation, competitive binding assays, and immunohistochemistry staining.

[0128] Protein M and its derivatives The protein M or a functional fragment or derivative thereof used in the use of the present invention may be from any mycobacterial species that produces a protein M that binds to an antibody. In some embodiments, the protein M or a functional fragment or derivative thereof is from Mycoplasma genitalium, Mycoplasma pneumoniae, or Mycoplasma penetrans.

[0129] In some embodiments, the protein M or a functional fragment or derivative thereof may be any of the protein M sequences described in PCT Publication No. 2014 / 014897 and U.S. Publication No. 2017 / 0320921, which are incorporated by reference in their entireties. In some embodiments, the 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., a fragment set forth in SEQ ID NO: 3 or a derivative thereof). In some embodiments, the 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., a fragment set forth in SEQ ID NO: 24 or a derivative thereof). In some embodiments, the protein M or a functional fragment or derivative thereof is Mycoplasma penetrans protein M or a functional fragment or derivative thereof. In some embodiments, the protein M or a functional fragment or derivative thereof is a protein M fragment or a derivative of a fragment thereof, e.g., a fragment that does not include the transmembrane domain and / or does not include the C-terminus, e.g., a functional fragment comprising, consisting essentially of, or consisting of, e.g., 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 the M. genitalium protein M (SEQ ID NO: 1), or equivalent residues from another protein M. The term "about" applied to each end of the recited fragment means that one or both of the terminal residues may vary slightly, e.g., by about 5, 4, 3, or 2 amino acids on either side of the recited residue. The equivalent residues from another protein M can be readily determined by one of skill in the art by performing a sequence alignment between the M. genitalium protein M and the other protein M. For example, Figure 27 shows a sequence alignment of amino acids 74 to 479 of wild-type M. genitalium protein M (SEQ ID NO: 3) with the equivalent 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) having an N-terminal 6-His tag followed by a thrombin cleavage site.

[0130] As used herein, the term "derivative" is used to refer 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 retains significant biological activity of protein M. Minor modifications include, but are not limited to, one or several amino acid side chain changes, one or several amino acid changes (including deletions, insertions, and / or substitutions), one or several atom stereochemical changes (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 20 or fewer amino acid residue mutations (deletions, insertions, and / or substitutions in any combination), such as 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 a functional fragment thereof.

[0131] In some embodiments, protein M or a functional fragment or derivative thereof may be modified for in vivo use by adding a blocking agent to the amino-terminus and / or carboxyl-terminus to promote survival of the relevant polypeptide in vivo. This may be useful in situations where the peptide termini are prone to degradation by proteases. Such blocking agents may include, but are not limited to, additional related or unrelated peptide sequences that may be coupled to the amino- and / or carboxyl-terminal residues of the administered protein. This may be done chemically during synthesis of the protein or by recombinant DNA techniques by 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, may 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 may be replaced with a different moiety. Similarly, the protein may be covalently or non-covalently coupled to a pharma-ceutically acceptable "carrier" protein prior to administration.

[0132] In one aspect of the invention, protein M derivatives are modified Mycoplasma protein M or functional fragments thereof that contain mutations that increase or at least maintain the thermal stability of protein M. These modified protein M derivatives have improved suitability for use in in vivo and other methods requiring high temperatures (e.g., about 37° C.) at which wild-type protein M may be denatured.

[0133] In some embodiments, a 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 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, 25.0°C, 30.0°C, or more, above the Tm of the wild-type protein M or functional fragment thereof. In some embodiments, the modified protein M or functional fragment thereof has a maintained (i.e., within 0.5°C) Tm compared to the Tm of the wild-type protein M or functional fragment thereof. The Tm may 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.

[0134] In some embodiments, the modified Mycoplasma protein M or functional fragments 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 fragments 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.

[0135] In some embodiments, the modified Mycoplasma protein M or a functional fragment thereof is derived from protein M of Mycoplasma genitalium, Mycoplasma pneumoniae, or Mycoplasma penetrans.

[0136] In some embodiments, the modified Mycoplasma protein M or functional fragment thereof is a fragment of 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) of the M. genitalium protein M (SEQ ID NO: 3), or the equivalent residues of the M. pneumoniae protein M (SEQ ID NO: 24).

[0137] In some embodiments, the one or more mutations are located in a portion of protein M known to affect thermostability. In some embodiments, the one or more mutations are not located in portions 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, 37 38, 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 equivalent 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 protein M of M. pneumoniae protein M (SEQ ID NO: 23) (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, 300, 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, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 4, 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 residues 469-479 of the M. genitalium protein M (SEQ ID NO: 1) or in the equivalent residues of the M. pneumoniae protein M (SEQ ID NO: 23).

[0138] The inventors have used computational analysis to identify residues in protein M that, when mutated, are predicted to increase or maintain the Tm of the protein. Thus, 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, 201, 202, 203, 204, 205, 206, 207, 20 5, 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, 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, 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 the equivalent residues of M. pneumoniae protein M (SEQ ID NO: 23), or any combination thereof. In some embodiments, the one or more mutations are those listed in Table 4, or any combination thereof. In some embodiments, the one or more mutations areResidues of M. pneumoniae protein M (SEQ ID NO: 23), or any combination thereof: 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, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 300, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337 54, 155, 156, 157, 158, 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, 8, 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, 285, 286, 287, 288, 289, 290, 291, 2 92, 293, 294, 295, 296, 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, 32 3, 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.

[0139] 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, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 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, 337, 339, 340, 341, 342, 343, 344, In some embodiments, the one or more mutations are at 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 the equivalent residues of M. pneumoniae protein M (SEQ ID NO: 23), or any combination thereof. In some embodiments, the one or more mutations are those listed in Table 5, or any combination thereof.

[0140] We have 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 give rise to modified protein M with even higher Tm (see Figure 15A).

[0141] Thus, in some embodiments, the one or more mutations are at a residue that has been demonstrated to increase the 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 of the M. genitalium protein M (SEQ ID NO: 1), or the equivalent residues of the M. pneumoniae protein M (SEQ ID NO: 23), or any combination thereof.

[0142] In some embodiments, the one or more mutations are at the following residues of 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 equivalent residue, or combination of residues, of M. pneumoniae protein M (SEQ ID NO: 23).

[0143] In some embodiments, the one or more mutations are in the 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 equivalent residues of M. pneumoniae protein M (SEQ ID NO: 23).

[0144] In some embodiments, the one or more mutations are at residues that have been demonstrated to maintain Tm, either alone or in combination with other mutations, for example, the one or more mutations are at residues 147, 150, 156, 225, 232, 245, 272, 276, 277, 279, 300, 310, 355, 378, 468 of the M. genitalium protein M (SEQ ID NO: 1), or the equivalent residues of the M. pneumoniae protein M (SEQ ID NO: 23), or any combination thereof.

[0145] In some embodiments, the one or more mutations are at the following residues of 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 at a residue selected from the equivalent residue, or combination of residues, of M. pneumoniae protein M (SEQ ID NO: 23).

[0146] In some embodiments, the one or more mutations are in the 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 equivalent residues of M. pneumoniae protein M (SEQ ID NO: 23).

[0147] In some embodiments, the one or more mutations are in the M. pneumoniae protein M (SEQ ID NO:23) a) A77E, b) K125R, c) V165Q, d) H170T, or e) A280V is selected from.

[0148] In some embodiments, the one or more mutations are in the M. genitalium protein M (SEQ ID NO:1). a) 159; b) 182; c) 102; d) 161; e)86; f)101; g) 147; h)236; i) 348; j)424; k)147; l)321; m)389; n)442; o)119; p)179; q) 186, or r)103 or the equivalent residues of M. pneumoniae protein M (SEQ ID NO: 23), or any combination thereof.

[0149] In some embodiments, the one or more mutations are in the M. genitalium protein M (SEQ ID NO:1). a) Q159C and A182C, b) A102C and T161C, or c) I86C and F101C or the equivalent residues of M. pneumoniae protein M (SEQ ID NO: 23), or any combination thereof, wherein the modified residues of a), b), or c) are capable of forming a disulfide bond between the modified residues.

[0150] In some embodiments, the one or more mutations are in the M. genitalium protein M (SEQ ID NO:1). a) H147Y, b) H236Y, c) H348Y, d) H424Y, or e) H147Q or the equivalent residue of M. pneumoniae protein M (SEQ ID NO: 23), or any combination thereof, and the mutation can prevent histidine protonation at that site at low pH, thereby preventing protein destabilization.

[0151] In some embodiments, the one or more mutations are in the M. genitalium protein M (SEQ ID NO:1). a) N321H, b) Y389H, c) N442H, d) P119H, e) T179H, f) G186H, or g) N103H or the equivalent residues of M. pneumoniae protein M (SEQ ID NO: 23), or any combination thereof, and the mutations can increase pH sensitivity and thereby improve elution conditions for antibody purification.

[0152] In some embodiments, the one or more mutations are at residues 155, 203, 243, 248, and 358 of the M. pneumoniae protein M (SEQ ID NO: 23).

[0153] In some embodiments, the one or more mutations are A155E, K203R, H243T, V248Q, and A358V of the M. pneumoniae protein M (SEQ ID NO: 23).

[0154] In some embodiments, the mutation is at residue 338 of the M. genitalium protein M (SEQ ID NO:1) (eg, Y388N).

[0155] The modified Mycoplasma protein M or functional fragments thereof may include further modifications other than mutations in the amino acid sequence. In some embodiments, one or more glycosylation sites in the protein M sequence are removed, for example, 1, 2, or 3 glycosylation sites. Three N-glycosylation sites are predicted in M. genitalium protein M based on both sequence and structure analysis using the NGlycPred server. These include N177, N213, and N274. Two O-glycosylation sites are predicted in M. genitalium protein M based on structure analysis using the NetOGlyc4.0 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 fragments thereof. Altering the glycosylation pattern may add to the thermal stability of the protein and / or may alter the immunogenicity of the protein by blocking antibody recognition.

[0156] For expression and purification purposes, the modified Mycoplasma protein M or functional fragment may contain a secretory peptide, for example at the N-terminus, so that the expressed protein can be secreted from the cell 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 in mammalian cells, and Sec or Tat in bacterial cells. The secretory peptide may or may not be removed from protein M before it is used in the methods of the invention.

[0157] 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 antibodies. The inventors have used computer analysis to identify residues in protein M that are predicted to increase the affinity of the protein for antibodies when mutated. Thus, 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 the M. genitalium protein M (SEQ ID NO: 1), or the equivalent residues of the M. pneumoniae protein M (SEQ ID NO: 23), or any combination thereof. 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 or near the residues of M. pneumoniae protein M (SEQ ID NO: 23), or any combination thereof 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, 300, 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, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 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.

[0158] In some embodiments, the modified Mycoplasma protein M or functional fragment may contain one or more additional mutations that modify the pH sensitivity of the protein, thereby altering the affinity of the protein for antibodies. Using computational analysis, the inventors have identified residues in 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 at the equivalent 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.

[0159] In some embodiments, the modified Mycoplasma protein M or functional fragment may contain one or more additional mutations that reduce or eliminate affinity for an antibody, including, but not limited to, mutations at residues 390 and 444, e.g., 390E and Y444K of the M. genitalium protein M (SEQ ID NO:1), or the equivalent residues of the M. pneumoniae protein M (SEQ ID NO:23).

[0160] In some embodiments, the modified Mycoplasma protein M or functional fragment may include a deletion of about residue 185 to about residue 342 of M. genitalium protein M (SEQ ID NO: 1), or the equivalent residues of another Mycoplasma protein M. In some embodiments, the deleted amino acids are replaced by a short linker. In some embodiments, the deletion results in newly exposed hydrophobic residues, and these newly exposed residues have one or more mutations.

[0161] In one aspect of the invention, the fusion protein comprises a modified Mycoplasma protein M or a functional fragment thereof, a linker, and a peptide. In some embodiments, the fusion protein comprises a modified Mycoplasma protein M or a functional fragment thereof with one or more mutations that enhance the affinity or avidity of the modified Mycoplasma protein M or a functional fragment thereof for an antibody. In some embodiments, the peptide is a dimerization peptide (e.g., IL-GCN4, C-IL-GCN4). In other embodiments, the peptide is a Fab binding polypeptide or an Fc binding polypeptide. In some embodiments, the Fab binding polypeptide is Protein L, Protein A, or Protein G. In some embodiments, the Fab binding polypeptide binds to the Fc portion of an immunoglobulin and / or reduces Fc receptor recycling of the immunoglobulin and / or increases binding of the fusion protein to the immunoglobulin. In other embodiments, the peptide comprises a dimerization peptide and Protein L. In one embodiment, the fusion protein comprises, in order, starting from the N-terminus, a dimerization peptide, a first linker, protein L, a second linker, and a modified Mycoplasma protein M or a functional fragment thereof.

[0162] In other embodiments, the peptide is an Fc binding polypeptide, which may be an Fc receptor polypeptide. In some embodiments, the peptide comprises an Fc binding polypeptide and protein L. In one embodiment, the fusion protein comprises, starting from the N-terminus, an Fc binding polypeptide, a first linker, protein L, a second linker, and a modified Mycoplasma protein M or a functional fragment thereof, in that order. In some embodiments, the peptide is an Fc domain and may comprise a complement binding residue for binding to an Fc receptor polypeptide, and / or one or more mutations of a complement binding residue for binding to an Fc receptor polypeptide. In some embodiments, the peptide is an Fc receptor protein, and the fusion protein reduces cellular uptake of antibodies, reduces antibody recycling, causes immunoglobulin depletion due to reduced antibody recycling, and / or increases the affinity of the fusion protein for immunoglobulins. In other embodiments, the fusion protein comprises two or more modified Mycoplasma protein M or a functional fragment thereof. In other embodiments, the fusion protein comprises a half-life extending moiety, e.g., human albumin or the addition of polyethylene glycol (PEG), a protein modification, e.g., acylation, methylation, phosphorylation, sulfation, farnesylation, ubiquitination, site-selective conjugation, glycosylation, introduction of PTMs, PEGylation, ligation, polymerization of protein-based initiators, or any modification that may alter binding, affinity, binding target, or any combination thereof.

[0163] The M protein of the present invention may be produced and characterized by methods well known in the art and described herein, for example by recombinant expression.

[0164] Further aspects of the invention provide isolated polynucleotides encoding protein M of the invention or a functional fragment or derivative thereof, and expression cassettes for producing protein M or a functional fragment or derivative thereof.

[0165] The polynucleotide may be operably linked to a regulatory element to aid in the 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).

[0166] In some embodiments, the polynucleotide may be codon-optimized to enhance expression of the protein 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. An example is the sequence of SEQ ID NO: 26, codon-optimized for expression in human cells, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto. 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. An example is the sequence of SEQ ID NO: 25, codon-optimized for expression in both E. coli and human cells, or a sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical thereto.

[0167] Another aspect of the present invention is a vector, e.g., an expression vector, comprising a polynucleotide of the present invention. The vector can be any type of vector known in the art, including, but not limited to, a plasmid vector and a viral vector. The vector can be, for example, a bacterial vector (e.g., an E. coli vector) or a mammalian cell vector (e.g., a human cell vector).

[0168] Further aspects of the invention relate to cells (e.g., isolated cells, transformed cells, recombinant cells, etc.) comprising the polynucleotides and / or vectors of the invention. Thus, various embodiments of the invention are directed to recombinant host cells comprising the vectors (e.g., expression cassettes). Such cells may be isolated cells. In some embodiments, the polynucleotide is stably integrated into the genome of the cell. In some embodiments, the cell may be a bacterial cell, such as E. coli, or a mammalian cell, such as a human cell.

[0169] A further aspect of the present invention relates to a kit comprising the modified Mycoplasma protein M or its functional fragment, polynucleotide, vector, and / or transformed cell of the present invention. The kit may include additional reagents for carrying out one of the methods described herein. The reagents may be included in a suitable package or container. The additional reagents include, but are not limited to, buffers, labels, enzymes, detection reagents, etc.

[0170] 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 long-term storage without losing the functionality of the active ingredients. The kit may further be provided with instruction materials. The instruction may be printed on paper or another substrate and / or provided as an electronically readable medium.

[0171] Different drugs As described above, the heterologous agent may be one against which neutralizing antibodies are present in the subject prior to administration of the heterologous agent, or one against which neutralizing antibodies may be generated upon administration to the subject. In some embodiments, the heterologous agent may be a nucleic acid delivery vector (e.g., a viral or non-viral vector), a gene editing complex (e.g., a CRISPR complex), a protein, or a nucleic acid.

[0172] Any nucleic acid sequence of interest can be delivered in the nucleic acid delivery vectors of the invention. Nucleic acids of interest include those that encode polypeptides, including therapeutic (e.g., for pharmaceutical or veterinary uses), immunogenic (e.g., for a vaccine), or diagnostic polypeptides.

[0173] Therapeutic polypeptides include cystic fibrosis transmembrane conductance regulator (CFTR), dystrophin (including mini-dystrophin and micro-dystrophin (see, 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 mutant, sarcospan, utrophin (Tinsley et al. 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-isopropyl ether, β ... interferon, beta-interferon, interferon-gamma, interleukin-2, interleukin-4, granulocyte-macrophage colony-stimulating factor, lymphotoxin, etc.), peptide growth factors, neurotrophic factors, and hormones (e.g., growth hormone, insulin, insulin-like growth factor 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-alpha and -beta, etc.), lysosomal acid alpha-glucosidase, alpha-galactosidase A, receptors (e.g., tumor necrosis growth factor alpha soluble receptor), S100A1, parvalbumin,Molecules that affect adenylyl cyclase type 6, G protein-coupled receptor kinase type 2 knockdown, such as 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, tumor necrosis factor), proteins that confer resistance to drugs used in cancer treatment, tumor suppressor gene products (e.g., p53, Rb, Wt-1), TRAIL, FAS ligand, and other polypeptides that have a therapeutic effect in a subject in need thereof. Parvovirus vectors can also be used to deliver monoclonal antibodies and antibody fragments, such as antibodies or antibody fragments against myostatin (see, e.g., Fang et al., Nature Biotechnol. 23:584-590 (2005)).

[0174] Nucleic acid sequences encoding a polypeptide 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.

[0175] 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), RNA that affects 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), interfering RNA (RNAi) including siRNA, shRNA, or miRNA that mediate 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 to Yuan et al.), and the like.Exemplary non-coding RNAs include RNAi against multidrug resistance (MDR) gene products (e.g., for the treatment and / or prevention of tumors and / or for administration to the heart to prevent chemotherapy damage), RNAi against myostatin (e.g., Duchenne muscular dystrophy), RNAi against VEGF (e.g., for the treatment and / or prevention of tumors), RNAi against phospholamban (e.g., for the treatment of 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 inhibitory or dominant negative molecules, such as phospholamban S16E (e.g., for the treatment of cardiovascular disease, see, e.g., Hoshijima et al., J. Gene Med. 10:132-142 (2008), and Li et al., Acta Pharmacol Sin. 26:51-55 (2005)). al. Nat. Med. 8:864-871 (2002)), RNAi against adenosine kinase (e.g., epilepsy), RNAi against sarcoglycans [e.g., alpha, beta, gamma], RNAi against myostatin, myostatin propeptide, follistatin, or activin type II soluble receptor, RNAi against anti-inflammatory polypeptides, e.g., I kappa B dominant mutant, and RNAi against pathogens and viruses (e.g., hepatitis B virus, human immunodeficiency virus, CMV, herpes simplex virus, human papilloma virus).

[0176] Alternatively, in certain embodiments of the invention, the nucleic acid may encode protein phosphatase inhibitor I (I-1), serca2a, a zinc finger protein regulating the phospholamban gene, Barkct, beta2-adrenergic receptor, beta2-adrenergic receptor kinase (BARK), phosphoinositide-3 kinase (PI3 kinase), a molecule that affects G protein-coupled receptor kinase type 2 knockdown, such as truncated constitutively active bARKct, calsarcin, RNAi against phospholamban, a phospholamban inhibitory molecule or a dominant negative molecule, such as phospholamban S16E, enos, inos, or a bone morphogenetic protein (including BNP2, 7, etc., RANKL and / or VEGF).

[0177] The nucleic acid delivery vector may also contain a nucleic acid that shares homology with and recombines with a locus on a host chromosome. This approach can be used, for example, to correct a genetic defect in the host cell.

[0178] The invention also provides nucleic acid delivery vectors expressing immunogenic polypeptides, e.g., for vaccination. The nucleic acid may encode any immunogen 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.

[0179] 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 to Young et al.; U.S. Patent No. 5,905,040 to Mazzara et al.; U.S. Patent No. 5,882,652 to Samulski et al.; U.S. Patent No. 5,863,541 to Samulski et al.). Antigens may be present within the parvovirus capsid. Alternatively, antigens may be expressed by nucleic acids introduced into the recombinant vector genome. Any immunogen of interest, as described herein and / or known in the art, may be provided by the nucleic acid delivery vector.

[0180] An 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, an immunogenic polypeptide can be an orthomyxovirus immunogen (e.g., an influenza virus immunogen, such as an influenza virus hemagglutinin (HA) surface protein, or an 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 the Lassa virus nucleocapsid protein and the Lassa virus envelope glycoprotein), a poxvirus immunogen (e.g., a vaccinia virus immunogen, such as the 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., 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.

[0181] Alternatively, the immunogenic polypeptide can be any tumor or cancer cell antigen. Optionally, the tumor or cancer antigen is expressed on the surface of a cancer cell. 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. Pat. 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 antigens (WO 90 / 05142); telomerase; nuclear matrix protein; 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 cancer, and other cancers or malignancies now known or later identified (see, e.g., Rosenberg, (1996) Ann. Rev. Med. 47:481-91).

[0182] One skilled in the art will understand that the nucleic acid of interest can be operably linked to appropriate control sequences. For example, the heterologous nucleic acid can be operably linked to expression control elements, such as transcriptional / translational control signals, origins of replication, polyadenylation signals, internal ribosome entry sites (IRES), promoters, and / or enhancers.

[0183] Those skilled in the art will appreciate that a variety of promoter / enhancer elements can be used depending on the level and tissue-specific expression desired. Promoters / enhancers can be constitutive or inducible depending on the expression pattern desired. Promoters / enhancers can be native or foreign and can be natural or synthetic sequences. By foreign it is meant that the transcription initiation region is not found in the wild-type host into which it is introduced.

[0184] In certain embodiments, the promoter / enhancer element may be native to the target cell or subject to be treated. In representative embodiments, the promoter / enhancer element may be native to the heterologous nucleic acid sequence. The promoter / enhancer element is generally selected to function in the target cell of interest. Moreover, in certain embodiments, the promoter / enhancer element is a mammalian promoter / enhancer element. The promoter / enhancer element may be constitutive or inducible.

[0185] Inducible expression control elements are typically advantageous in applications where it is desirable to provide regulation over the expression of a nucleic acid sequence. Inducible promoter / enhancer elements for gene delivery may 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, Tet on / off elements, RU486-inducible promoter, ecdysone-inducible promoter, rapamycin-inducible promoter, and metallothionein promoter.

[0186] In embodiments in which the nucleic acid sequence is 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.

[0187] 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 a cell in vitro, for example for ex vivo gene therapy. Nucleic acid delivery vectors are further useful in methods for delivering a nucleic acid 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 a subject. The subject may need the polypeptide because of a deficiency of the polypeptide. Furthermore, the method can be carried out because the production of the polypeptide or functional RNA in a subject may provide some beneficial effect.

[0188] 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 a screening method).

[0189] In general, the nucleic acid delivery vectors of the 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 disease states include, but are not limited to, cystic fibrosis (cystic fibrosis transmembrane conductance regulator) and other lung diseases, hemophilia A (factor VIII), hemophilia B (factor IX), thalassemia (β-globin), anemia (erythropoietin) and other blood diseases, Alzheimer's disease (GDF; neprilysin), multiple sclerosis (β-interferon), Parkinson's disease (glial cell line-derived neurotrophic factor [GDNF]), Huntington's disease (repeat removal by RNAi), amyotrophic lateral sclerosis, epilepsy (Galanthus glomerulosa), and other conditions in which it is beneficial to deliver a therapeutic polypeptide or functional nucleic acid. ranin, neurotrophic factors), other neurological disorders, cancer (cytokines including endostatin, angiostatin, TRAIL, FAS ligand, interferons; RNAi including RNAi against VEGF or multidrug resistance gene products), diabetes (insulin), Duchenne (dystrophin, mini-dystrophin, insulin-like growth factor I, sarcoglycans [e.g., alpha, beta, gamma]), RNAi against myostatin, myostatin propeptide, follistatin, activin type II soluble receptor , anti-inflammatory polypeptides, e.g., I kappa B dominant mutations, sarcospan, utrophin, mini-utrophin, induction of exon skipping by RNAi against splice junctions of the dystrophin gene [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 muscular dystrophies including skeletal muscle dystrophies, 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 disorders, 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 (other diseases of the eye and retina, e.g., 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, congestive heart failure or Molecules that affect the heart, including peripheral arterial disease (PAD), such as protein phosphatase inhibitor I (I-1), serca2a, zinc finger protein regulating the phospholamban gene, Barkct, β2-adrenergic receptor, β2-adrenergic receptor kinase (BARK), phosphoinositide-3 kinase (PI3 kinase), S100A1, parvalbumin, adenylyl cyclase type 6, and G protein-coupled receptor kinase type 2 knockdown, such as truncated constructs and / or phospholamban S16E), arthritis (insulin-like growth factor), joint disorders (insulin-like growth factor 1 and / or 2), intimal hyperplasia (e.g., by delivering enos, inos), improved survival of heart transplants (superoxide dismutase), AIDS (soluble CD4), muscle wasting (insulin-like growth factor I), renal deficiency (erythropoietin), anemia (erythropoietin), arthritis (anti-inflammatory factors, e.g., IRAP and TNFα soluble receptor), hepatitis (α-interferon), LDL receptor deficiency (LDL receptor), hyperammonemia (ornithine transcarbamylase), Krabbe disease (galactocerebrosidase), Batten disease, spinal cord ataxias including SCA1, SCA2, and SCA3, phenylketonuria (phenylalanine hydroxylase), autoimmune diseases, etc. The present invention further relates to a method for treating organ transplantation, which may be used to increase the success of transplantation and / or to decrease the negative side effects of organ transplantation or as an adjunct therapy, 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, following fracture or surgical removal in cancer patients.

[0190] Gene transfer has substantial potential applications for understanding and providing therapy for pathologies. There are many genetic diseases for which the defective genes are known and have been cloned. In general, the pathologies are divided into two classes: deficiency states, which are generally recessively inherited and usually enzyme deficiency states, and disequilibrium states, which are typically dominantly inherited and may involve regulatory or structural proteins. For diseases of deficiency states, gene transfer can be used to carry normal genes to affected tissues for replacement therapy and to create animal models for the disease using antisense mutations. For disequilibrium pathologies, gene transfer can be used to create pathologies in model systems, which can then be used in attempts to combat the pathology. Thus, nucleic acid delivery vectors allow for the treatment and / or prevention of genetic diseases.

[0191] 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 a specific target protein by the cells. Thus, functional nucleic acids can be administered to subjects in need thereof to reduce the expression of a specific protein.

[0192] 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.

[0193] Nucleic acid delivery vectors can also be used for a variety of non-therapeutic purposes, including, but not limited to, use in protocols for evaluating gene targeting, clearance, transcription, translation, etc., as will be apparent to those skilled in the art. Nucleic acid delivery vectors can also be used for the purpose of evaluating safety (spread, toxicity, immunogenicity, etc.). Such data are taken into account, for example, by the U.S. Food and Drug Administration as part of the regulatory approval process prior to evaluation of clinical efficacy.

[0194] 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 to initiate an active immune response by the subject against the immunogenic polypeptide. The immunogenic polypeptide is as described above. In some embodiments, a protective immune response is elicited.

[0195] Alternatively, the nucleic acid delivery vector may be administered to cells ex vivo, and the modified cells are 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 is expressed, and an immune response to the immunogen can be induced in the subject. In certain embodiments, the cells are antigen-presenting cells (e.g., dendritic cells).

[0196] "Active immune response" or "active immunity" is characterized by "the participation of host tissues and cells after encounter with an immunogen. It involves the differentiation and proliferation of immunocompetent cells in lymphoreticular tissue, resulting in the synthesis of antibodies or the development of cell-mediated reactivity, 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 acquired by "the transfer of preformed substances (antibodies, transfer factors, thymus grafts, interleukin-2) from an actively immunized host to a non-immunized host." Id.

[0197] As used herein, a "protective" immune response or "protective" immunity indicates that the immune response confers 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 thereof.

[0198] 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.

[0199] The nucleic acid delivery vector can also be administered for cancer immunotherapy by administering a nucleic acid delivery vector that expresses one or more cancer cell antigens (or immunologically similar molecules) or any other immunogen that generates an immune response against cancer cells. To explain, for example, to treat a patient with cancer and / or to 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 that includes a nucleic acid that encodes a cancer cell antigen. The nucleic acid delivery vector can be administered to a subject by 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.

[0200] 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.

[0201] As used herein, the term "cancer" includes tumor-forming cancers. Similarly, the term "cancerous tissue" includes tumors. "Cancer cell antigens" includes tumor antigens.

[0202] The term "cancer" has its art-understood meaning, e.g., the uncontrolled proliferation of cells with 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 tumors, and any other cancer or malignant condition now known or later identified. In an exemplary embodiment, the present invention provides a method of treating and / or preventing tumor-forming cancer.

[0203] The term "tumor" is also understood in the art as, for example, an abnormal mass of undifferentiated cells within a multicellular organism. Tumors can be malignant or benign. In representative embodiments, the methods disclosed herein are used to prevent and treat malignant tumors.

[0204] 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 the prevention or reduction or at least partial elimination of metastasis of cancer, and / or the prevention or reduction or at least partial elimination of the growth of metastatic nodules.

[0205] The term "cancer prevention" or "preventing cancer" and equivalent terms contemplate that the method at least partially eliminates, or reduces and / or delays the incidence and / or severity of cancer onset. In other words, the onset of cancer in a subject is reduced in chance or probability and / or delayed.

[0206] 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 inducing 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).

[0207] 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 chemoattractant protein-1, granulocyte-macrophage colony-stimulating factor, and lymphotoxin). Thus, an immunomodulatory cytokine (preferably a CTL-inducing cytokine) can be administered to a subject in combination with a viral vector.

[0208] The cytokine may be administered by any method known in the art. Exogenous cytokines may be administered to a subject, or a nucleic acid encoding the cytokine may be delivered to a subject using a suitable vector and the cytokine produced in vivo.

[0209] Subjects, Pharmaceutical Formulations, and Modes of Administration The methods of the invention find use in both veterinary and medical applications. Suitable subjects include birds, reptiles, amphibians, fish, and mammals. The term "mammal" as used herein 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 neonates, infants, adolescents, and adults. Optionally, the subject is "in need" of the methods of the invention, for example, because the subject is believed to have or 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 a human.

[0210] 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 as possible in the subject's life, 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, for example, after the disease or disorder is identified 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, for example, after the disease or disorder is identified 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.

[0211] 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 heterologous agent, a pharma- ceutically acceptable carrier, and optionally other medicinal agents, pharmaceutical agents, stabilizers, buffers, carriers, adjuvants, diluents, etc. For injection, the carrier is usually a liquid. For other modes of administration, the carrier can be either a solid or liquid. For administration by inhalation, the carrier is inhalable and can be in solid or liquid particulate form.

[0212] "Pharmaceutically acceptable" means a substance that may be administered to a subject without causing toxic or other undesirable effects, ie, any undesired biological effects.

[0213] One aspect of the present invention is a method of introducing a nucleic acid into a cell 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 the cell in an appropriate amount, e.g., a multiplicity of infection, according to standard transduction methods suitable for the particular target cell. The titer of the viral vector 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.

[0214] The cells 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, cells of the eye (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), liver cells, 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, and the like. In representative embodiments, the cells can be any progenitor cells. As a further possibility, the cells can be stem cells (e.g., neural stem cells, hepatic stem cells). As a further alternative, the cells can be cancer or tumor cells. Furthermore, the cells may be from any species of origin, as described above.

[0215] 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 therein, 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, for example, 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).

[0216] Cells suitable for ex vivo gene delivery are described above. The dosage of cells to be administered to a subject will vary 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 6The cells are administered per dose in a pharma- ceutically acceptable carrier. In certain embodiments, the 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.

[0217] In some embodiments, the nucleic acid delivery vector is introduced into a cell, and the cell can be administered to a subject to induce an immunogenic response against the delivered polypeptide (e.g., expressed as a transgene or within a capsid). Typically, a quantity of cells expressing an immunogenically effective amount of the polypeptide is administered in combination with a pharma- ceutically acceptable carrier. An "immunogenically effective amount" is an amount of expressed polypeptide sufficient to induce an active immune response against the polypeptide in the subject to whom the pharmaceutical formulation 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 thereof.

[0218] A further aspect of the present invention is a method of administering a heterologous agent (e.g., a nucleic acid delivery vector) to a subject. The administration of the nucleic acid delivery vector to a human subject or animal in need thereof can be by any means known in the art. Optionally, the nucleic acid delivery vector is delivered in a therapeutically or prophylactically effective amount in a pharma- ceutically acceptable carrier.

[0219] The nucleic acid delivery vector can further be administered (e.g., as a vaccine) to induce an immunogenic response. Typically, the immunogenic composition of the present invention comprises an immunogenically effective amount of the nucleic acid delivery vector in combination with a pharma- ceutically acceptable carrier. Optionally, the dosage is an amount sufficient to generate a protective immune response (as defined above). The degree of protection provided need not be complete or permanent, so long as the benefits of administering the immunogenic polypeptide outweigh any disadvantages thereof. The subject and immunogen are as described above.

[0220] 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 being treated and / or prevented, the condition of the individual subject, the particular nucleic acid delivery vector, and the nucleic acid being delivered, and can be determined in a routine manner. Exemplary doses for achieving 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 Transducing units, optionally about 10 8 ~10 13 The titer of transducing units.

[0221] 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 a desired level of gene expression.

[0222] Exemplary modes of administration include oral, rectal, transmucosal, intranasal, inhalation (e.g., by aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intraendothelial, intrauterine (or intraembryo), 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 airway 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).

[0223] Administration can be at any site in a subject, including, but not limited to, a site selected from the group consisting of brain, skeletal muscle, smooth muscle, heart, diaphragm, airway epithelium, liver, kidney, spleen, pancreas, skin, and eye.

[0224] Administration may be to the tumor (e.g., in 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.

[0225] 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 legs, 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 hallucis, abductor metatarsus, abductor pollicis brevis, adductor pollicis longus, adductor pollicis brevis, adductor hallucis, adductor pollicis longus, adductor magnus, adductor pollicis, anconeus, anterior scalene, genu articularis, biceps brachii, biceps femoris, brachialis, brachioradialis, buccinator, coracobrachialis, corrugator superior, deltoid, depressor anguli oris, depressor labii inferior, digastric, dorsal interosseous (hand), dorsal interosseous (foot), and extensor carpi radialis brevis. , extensor carpi radialis longus, extensor carpi ulnaris, extensor digitorum minimi, extensor digitorum brevis, extensor digitorum longus, extensor hallucis brevis, extensor hallucis longus, extensor indicis, extensor pollicis brevis, extensor pollicis longus, flexor carpi radialis, flexor carpi ulnaris, flexor digitorum brevis (of the hand), flexor digitorum brevis (of the 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, gastrocnemius, geniohyoid, gluteus maximus, gluteus medius, gluteus minimus, gracilis, iliocostalis cervicalis, iliocostalis lumborum, iliocostalis thoracicus, ilium muscles, inferior gemellus, inferior oblique, inferior rectus, infraspinatus, interspinatus, intertransverse, lateral pterygoid, lateral rectus, latissimus dorsi, levator anguli oris, levator labii superioris, levator palpebrae superioris, levator scapulae, rotator longus, longissimus capitis, longissimus cervicalis, longissimus thoracis, longus capitis, longus colli, medius (of the hand), medius (of the foot), masseter, medial pterygoid, medial rectus, middle scalene, multifidus, mylohyoid, oblique capitis inferior, oblique capitis superior, obturator externus, obturator internus, occipital, hemihyoid, opposite little finger, opposite thumb, orbicularis oculi, Orbicularis oris, Palmar interosseus, Palmaris brevis, Palmaris longus, Pubicius, Pectoralis major, Pectoralis minor, Peroneus brevis, Peroneus longus, Peroneus tertius, Piriformis, Plantar interosseus, Plantar, Platysma, Popliteus, Scalene posterior, 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, Luccius, Sartorius, Scalene minor, Semimembranosus, Semispinalis capitis, Semispinalis cervix, Semispinalis thoracic, Semitendinosus, Serratus anterior, Short rotator rotator), soleus, spinalis capitis, spinalis cervicis, thoracic spinalis, splenius capitis, splenius cervicis, sternocleidomastoid, sternohyoid, sternothyroid, stylohyoid, subclavius, subscapularis, superior gemellus, superior oblique, superior rectus, supinator, supraspinatus, temporalis, tensor fasciae latae, teres major, teres minor, pectoralis, thyrohyoid, tibialis anterior, tibialis posterior, trapezius, triceps brachii, vastus intermedius, vastus lateralis, vastus medialis, zygomaticus major, and zygomaticus minor, as well as any other suitable skeletal muscles known in the art.

[0226] Heterologous agents can be delivered to skeletal muscle by intravenous administration, intraarterial administration, intraperitoneal administration, limb perfusion (optionally 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, the heterologous agent is administered to a limb (arm and / or leg) of a subject (e.g., a subject with a muscular dystrophy such as DMD) by limb perfusion, optionally isolated limb perfusion (e.g., intravenously or intravenously). In embodiments of the present invention, the heterologous agent may be advantageously administered without the use of "hydrodynamic" techniques. Tissue delivery (e.g., to muscle) of vectors in the prior art is often enhanced by hydrodynamic techniques (e.g., intravenous / bolus intravenous administration), which increases pressure within the vasculature and promotes the ability of the factor to cross the endothelial cell barrier. In certain embodiments, the heterologous agent may be administered in the absence of hydrodynamic techniques, such as bolus injection and / or elevation of intravascular pressure (e.g., above normal systolic pressure, e.g., a 5%, 10%, 15%, 20%, 25% or less increase in intravascular pressure 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.

[0227] Administration to the myocardium includes administration to the left atrium, right atrium, left ventricle, right ventricle, and / or septum. Heterologous agents 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.

[0228] Administration to the diaphragm muscle can be by any suitable method, including intravenous administration, intraarterial administration, and / or intraperitoneal administration.

[0229] Administration to smooth muscle can be by any suitable method, including intravenous, intraarterial, and / or intraperitoneal administration, hi one embodiment, administration can be to endothelial cells located within, near, and / or on smooth muscle.

[0230] Delivery to the target tissue can also be accomplished by delivering a depot containing a heterologous agent. In an exemplary embodiment, a depot containing a heterologous agent can be 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. Pat. No. 7,201,898.

[0231] 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 cardiac disease (eg, PAD or congestive heart failure)).

[0232] In an exemplary embodiment, the present invention is used to treat and / or prevent disorders of skeletal, cardiac and / or diaphragm muscles.

[0233] In an exemplary embodiment, the invention provides a method of 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, the heterologous agent comprising dystrophin, mini-dystrophin, micro-dystrophin, myostatin propeptide, follistatin, activin type II soluble receptor, IGF-1, an anti-inflammatory polypeptide, such as I kappa B 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 may be administered to skeletal muscle, diaphragm muscle, and / or cardiac muscle, as described elsewhere herein.

[0234] Alternatively, the invention can be implemented to deliver nucleic acids to skeletal, cardiac or diaphragm muscles to be used as a platform for the production of normally circulating polypeptides (e.g., enzymes) or functional nucleic acids (e.g., functional RNAs, e.g., RNAi, microRNAs, antisense RNAs) for systemic delivery to blood or other tissues to treat and / or prevent disorders (e.g., metabolic disorders such as diabetes (e.g., insulin), hemophilia (e.g., factor IX or factor VIII), mucopolysaccharidoses (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 alpha-glucosidase] or Fabry disease [alpha-galactosidase A]) or glycogen storage diseases (e.g. Pompe disease [lysosomal acid alpha-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 US Patent Publication No. 2002 / 0192189.

[0235] Thus, in one aspect, the present invention encompasses a method of 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 a skeletal muscle of the subject), the heterologous agent comprising a nucleic acid encoding a polypeptide, and the metabolic disorder being 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 engineered to be secreted, e.g., by operative association with a secretion signal sequence known in the art). Without being limited to any particular theory of the invention, according to this embodiment, administration to a skeletal muscle can result in the secretion of the polypeptide into the systemic circulation and delivery to a target tissue. Methods of delivering a heterologous agent to a skeletal muscle are described in more detail herein.

[0236] The invention can also be practiced to produce antisense RNA, RNAi or other functional RNA (eg, ribozymes) for systemic delivery.

[0237] The present invention also relates to 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, the heterologous agent being, for example, an agent that inhibits sarcoplasmic endoreticulum Ca2+ receptor activator (SARCA) receptor activator (CR ... 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 (βARKct), pro Methods are provided that include nucleic acids encoding inhibitor of tein phosphatase 1 1, S100A1, parvalbumin, adenylyl cyclase type 6, molecules that affect G protein-coupled receptor kinase type 2 knockdown, such as, for example, 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.

[0238] Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Alternatively, heterologous agents may be administered in a local rather than systemic manner, for example in a depot or sustained release formulation. Additionally, heterologous agents can be delivered attached to a surgically implantable matrix (e.g., as described in U.S. Patent Publication No. 2004-0013645).

[0239] The heterologous agents disclosed herein can be administered to the lungs of a subject by any suitable means, optionally by administering an aerosol suspension of inhalable particles 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, for example, using a pressure-driven aerosol nebulizer or an ultrasonic nebulizer, as known to those skilled in the art. See, for example, U.S. Pat. No. 4,501,729. Aerosols of solid particles containing the heterologous agent can be similarly produced using any solid particulate drug aerosol generator, by techniques known in the pharmaceutical arts.

[0240] Heterologous agents can be administered to tissues of the CNS (eg, brain, eye), which may advantageously result in a broader distribution of the heterologous agent than would be observed in the absence of the present invention.

[0241] 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, psychiatric disorders including mood disorders (e.g., depression, bipolar disorder, persistent affective disorder, and chronic obstructive pulmonary disease). disorders, 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).

[0242] Disorders of the CNS include ocular disorders involving the retina, posterior tract, and optic nerve (eg, retinitis pigmentosa, diabetic retinopathy and other retinal degenerative diseases, uveitis, age-related macular degeneration, glaucoma).

[0243] 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.

[0244] Diabetic retinopathy, for example, is characterized by angiogenesis. Diabetic retinopathy can be treated by delivering one or more antiangiogenic factors intraocularly (e.g., intravitreally) or periocularly (e.g., sub-Tenon's area). One or more neurotrophic factors can also be co-delivered either intraocularly (e.g., intravitreally) or periocularly.

[0245] Uveitis is associated with inflammation. One or more anti-inflammatory factors may be administered by intraocular (e.g., intravitreal or anterior chamber) administration of a delivery vector of the invention.

[0246] Retinitis pigmentosa is, by comparison, characterized by retinal degeneration. In a representative embodiment, retinitis pigmentosa can be treated by intraocular (e.g., intravitreal) administration of a heterologous agent encoding one or more neurotrophic factors.

[0247] 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 region).

[0248] Glaucoma is characterized by increased intraocular pressure and loss of retinal ganglion cells. Treatment for glaucoma involves administering one or more neuroprotective agents that protect cells from excitotoxic damage using heterologous drugs. Such drugs include N-methyl-D-aspartate (NMDA) antagonists, cytokines, and neurotrophic factors that are delivered intraocularly, optionally intravitreally.

[0249] In other embodiments, the present invention may be used to treat seizures, for example, to reduce the onset, incidence or severity of seizures. The effectiveness of treatment against seizures can be assessed by behavior (e.g., tremors, eye or mouth tics) and / or electrographic measures (most seizures have characteristic electrographic abnormalities). Thus, the present invention may also be used to treat epilepsy characterized by multiple seizures over time.

[0250] In one representative embodiment, somatostatin (or its active fragments) is administered to the brain using the heterologous agents of the present invention to treat pituitary tumors. According to this embodiment, a heterologous agent encoding somatostatin (or its active fragments) 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.

[0251] In certain embodiments, the heterologous agent can include a secretion signal as described in US Pat. No. 7,071,172.

[0252] In an exemplary embodiment of the invention, the heterologous agent is administered to the CNS (e.g., 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.

[0253] Heterologous agents can be delivered to the cerebrospinal fluid (e.g., by lumbar puncture) for more distributed 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 tumors or cerebral infarction).

[0254] Heterologous agents can be administered to the desired region 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, as well as intramuscular delivery with retrograde delivery to motor neurons.

[0255] In certain embodiments, the heterologous agent is administered in a liquid formulation by direct injection (e.g., stereotactic injection) to the desired area or compartment within the CNS. In other embodiments, the heterologous agent may be provided by topical application to the desired area or by intranasal administration of an aerosol formulation. Administration to the eye may be by topical application of liquid drops. As a further alternative, the heterologous agent may be administered as a solid sustained release formulation (see, e.g., U.S. Pat. No. 7,201,898).

[0256] 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 where they can be transported to neurons.

[0257] Protein M or a functional fragment or derivative thereof may be administered by any route or schedule described above for the heterologous agent, or Protein M or a functional fragment or derivative thereof may be administered by a different route or schedule than the heterologous agent.

[0258] Having described the invention, the same will be described in more detail in the following examples, which are included herein for illustrative purposes only and are not intended as limitations upon the invention. EXAMPLES

[0259] Example 1: Methods AAV virus production: AAV vectors were produced using a standard approach of transfecting three plasmids into HEK293 cells. Briefly, the AAV transgene plasmid pTR-CBA-Luc was co-transfected 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 by anion exchange Q-column. The purified AAV vectors were titered by qPCR using primers aimed at amplifying segments of the packaged AAV transgene.

[0260] Protein production: Plasmid pET-28b(+), encoding protein M, a truncated M. genitalium protein MG281 lacking the transmembrane domain (amino acids 74-479), with an N-terminal His-Tag and a thrombin cleavage site, was provided by Rajesh. The plasmid was grown in electrocompetent DH10B cells and purified using the PureLink Maxi-Prep kit by 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 1-4 L culture volumes. The culture was then pelleted by centrifugation and frozen at -80°C.

[0261] Protein purification: Frozen bacterial cell culture pellets were thawed, lysed by sonication, DNAse treated, and clarified by centrifugation. The clarified bacterial lysate was dialyzed in nickel binding buffer (20 mM imidazole, 50 mM sodium phosphate, pH 7.4, 500 mM NaCl, 0.02% sodium azide) and passed over 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 over an S-100 size exclusion column, dialyzed into phosphate buffered saline with 2% glycerol, and quantified using spectrophotometry. The identity of Protein M was confirmed using SDS-PAGE protein gel electrophoresis for protein separation followed by Coomassie blue protein staining to correctly identify the 48 kDa Protein M band.

[0262] Western blot: After protein separation on 8% SDS-PAGE gel, proteins were transferred onto PVDF membrane. Immunoblotting was performed with anti-His primary antibody 1:1000 dilution (10 μg / ml) in 5% nonfat milk. Secondary goat anti-human IgG antibody was conjugated with horseradish peroxidase (1:10,000 dilution).

[0263] 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.

[0264] Human IVIG and immunized mouse serum: 10% human IVIG (Gamunex) was purchased from Grifols Therapeutics Inc. (Research Triangle Park, NC, USA). 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 boost of the same vector 2 weeks later and a second boost 6 weeks after the first, serum was collected and pooled. Human IVIG and mouse serum were aliquoted and stored at -80°C for further use.

[0265] In vitro AAV neutralization assay: NAb analysis was performed as previously described with minor modifications. Cells were pelleted by centrifugation, resuspended in serum-free X-Vivo10 medium, and then plated in either 48- or 96-well plate formats. Human IVIG or serum was serially diluted either 2-fold or 10-fold. AAV-Luc was incubated with either human IVIG or mouse serum for 1 h at 4° C., followed by addition of AAV and an additional 1 h incubation at 4° C. The AAV+serum culture was then mixed with cells suspended in serum-free medium at the time of plating. For neutralization experiments with protein M, three different incubation scenarios were tested at each step for 1 h at 4° C.: neutralizing serum was first incubated with protein M and then incubated with AAV, AAV was first incubated with neutralizing serum and then incubated with protein M, and AAV was first incubated with protein M and then incubated 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 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 luciferase signals were measured in a Wallac 1420 Victor2 automated plate reader. NAb titers were defined as the highest dilution at which luciferase activity was 50% lower than serum-free controls.

[0266] In vivo AAV neutralization and passive transfer of NAb serum: Different amounts of serum containing AAV NAbs were injected into the retro-orbital vein of C57BL / 6 mice 20 min prior to AAV injection. For mice that received protein M treatment prior to AAV administration, protein M (either 6.3 mg at a 2:1 ratio, 3.15 mg at a 1:1 ratio, or 1.58 mg at a 0.5:1 ratio) was delivered via the retro-orbital vein 5–15 min after serum injection. AAV was delivered at 2 × 10 12AAV-Luc vector was administered 5 min later by systemic injection of particles / kg. Imaging was performed 1 day after AAV administration, and at 1 week and 9 days after injection.

[0267] Example 2: Protein M counteracts the inhibitory activity of IVIG on AAV transduction To test the ability of protein M to block antibodies, an in vitro neutralization assay of AAV with human intravenous immunoglobulin gamma (IVIG) was set up. AAV neutralization assays were performed using serial dilutions of IVIG to determine the amount of IVIG that neutralized a given amount of AAV2. Luciferase activity generated by cell transduction with 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 proceed with the experimental design to test protein M blocking of IVIG on AAV neutralization. Next, a dose dilution ratio of protein M (SEQ ID NO:2) was performed by removing the His tag by thrombin cleavage, which was capable of blocking 12.5 μg of IVIG (Figure 2), and protein M was incubated with IVIG for 1 h and then with AAV2 for 1 h at 4°C before transduction in cell culture. A molar ratio of 2 molecules of protein M to 1 molecule of IgG was found to be sufficient to prevent neutralization to a level comparable to the no IVIG control. Furthermore, a higher molar ratio of protein M to IVIG was found to enhance the luciferase signal to a level higher than the no IVIG control (Figure 2). 8 molecules of protein M to 1 molecule of IgG increased luciferase expression by 2-fold. To see if higher molar ratios would further increase the enhancement, 8:1 and 20:1 ratios of protein M to IVIG were tested in the same experimental conditions. It was found that increasing the dose of Protein M by 2.5-fold (from 8:1 to 20:1 ratio) provided little further enhancement, as the corresponding signal was only increased by 0.25-fold over 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, and therefore Protein M without cleavage of the His-tag was used in subsequent experiments.

[0268] 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 8It was found that the dilution of protein M below 2 μg with respect to viral particles was lost. The equivalent molar ratio of protein M molecules to AAV particles below 40,000:1 at which enhancement was lost. Next, it was shown that the enhancement of transduction by protein M depends on the incubation of protein M with AAV. In FIG. 5, a transduction assay of cell cultures was performed in which protein M was added to AAV 1 hour before addition to the cells (pre-incubation, -1 hour time point), added at the time of transduction without pre-incubation (near transduction, 0 hour time point), or added 18 hours after AAV was added to the cells (post-transduction, 18 hour time point). It was found that a dose-dependent enhancement was seen in the pre-incubation group as in FIG. 4, but not in the other two groups in which protein M and AAV2 were not incubated together before the assay. Furthermore, the results also 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 the cells at the time of transduction had no effect on the luciferase signal. Finally, it was shown that protein M is unable to block neutralization when IVIG is first preincubated with AAV2 for 1 h and then incubated with protein M for 1 h, and that enhancement of AAV2 luciferase signal by protein M occurs only if AAV2 is not completely neutralized by IVIG. For this assay, 12.5 μg IVIG (neutralizing 75-80% of AAV2), 50 μg IVIG (neutralizing 99% of AAV2, Figure 1), or 200 μg IVIG (neutralizing 100% of AAV2) were used. In Figure 6, it was 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.

[0269] Example 4: Pre-incubation of AAV vector virions with protein M protects IVIG neutralization of AAV Next, instead of first interacting protein M with IVIG, we decided to test the effect of first pre-incubating protein M with AAV and then incubating with IVIG. We tested the ability of protein M to block neutralization when it was incubated with AAV2 for 1 h, followed by the addition of IVIG and another 1 h at 4°C before cell transduction. In Figure 7, it was found that by keeping the protein M concentration constant (8.25 μg) and serially diluting the IVIG dose from 50 μg to 3.12 μg (protein M to IVIG molar ratios from 1:2 to 8:1), protection of the vector from neutralization was achieved at a ratio of 2:1 or higher, similar to previous results when protein M was incubated with IVIG before the addition of AAV. In an attempt to simulate an environment closer to the in vivo situation where other types of proteins other than IgG are present in serum, a similar in vitro neutralization study was 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. 25 μg of IVIG or serum-free medium was then spiked into half of the wells to neutralize AAV or to serve as a control with no neutralizing activity. In Figure 8, when 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 indicate that protein M is capable of binding and blocking immunoglobulins even in the presence of other serum proteins.

[0270] Example 5: Protein M efficiently blocks the neutralizing activity of mouse serum against AAV in vitro To translate the findings from IVIG into a real-world context, in vitro neutralization experiments were first performed with the AAV8-luciferase vector using serum from mice immunized with the AAV8 capsid vector. When both serum from immunized mice and protein M were serially diluted to maintain a 2:1 molar ratio, results were compared with serum-only controls, revealing that protein M confers escape from neutralization for a neutralizing serum titer of 1:2,564, an estimated 100-fold dilution (50% neutralization with 0.0039 μl serum vs. 0.2744 μl serum in the presence of protein M, FIG. 9).

[0271] Example 6: Protein M efficiently blocks the neutralizing activity of mouse 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 10 100 μl of AAV8-NAb serum (titer 1:2,564) was administered to mice. Figure 10 shows that an approximate molar ratio of 2:1 (6.3 mg) of protein M relative to total immunoglobulin in mice can prevent neutralization of AAV8 by 1 μl of AAV8-NAb serum (titer 1:2,564). This is compared to in vivo neutralization assays of serially diluted anti-AAV8 serum, in which greater than 50% of AAV8-Luc was neutralized with serum volumes ranging from 1 μl to 0.001 μl, representing AAV NAb escape over a 1,000-fold concentration difference (Figure 11).

[0272] Example 7: Stability of Protein M / immunoglobulin complexes To test how stable the complexes of immunoglobulins and protein M are, an assay was first performed in vitro. Protein M was pre-incubated with mouse serum at a 2:1 molecular ratio for different durations from 1 h to 72 h at 37°C, and then AAV8 vectors were added for another hour at 4°C for neutralization analysis. As shown in Figure 12, the pre-formed 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 only PBS or medium. This result suggests that the complexes formed between protein M and immunoglobulins are very stable in vitro.

[0273] We next investigated the in vivo stability of Protein M after passive transfer of 0.3 μl of AAV8 neutralizing serum. If Protein M was administered and 3 hours were 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 IL-18 was found to be reduced by approximately 70% (FIG. 13). This result indicates that the NAb blocking effect of Protein M is short-term in vivo.

[0274] Gene therapy using AAV vectors has been successful in clinical trials. However, the high occurrence of AAV neutralizing antibodies in the human population prevents more patients from benefiting from this effective gene delivery. In this study, it was found that mycoplasma-derived protein M is capable of interacting with NAb to enhance AAV transduction. The minimum dose of protein M required to block NAb activity was two molecules of immunoglobulin. Direct interaction of AAV virions with protein M also increased AAV transduction. Neutralizing antibody assays showed that NAb activity was reduced approximately 100-fold when AAV-immunized mouse serum was incubated with protein M in vitro, and more than 1000-fold protection was observed in mice adoptively transferred with NAb-positive serum when protein M was applied. Although the immunoglobulin-protein M complex was stable over time in vitro, protein M gradually loses its protective function from immunoglobulin neutralization in vivo.

[0275] To overcome AAV NAbs, multiple strategies have been utilized in the laboratory. One approach is to mask the AAV surface to block Nab recognition, using polymers or exosomes for coating. Although this approach is promising, it may alter the AAV transduction profile. A second approach is to generate libraries of AAV capsid variants using error-prone PCR or DNA shuffling and select NAb escape mutants in vitro and in vivo in the presence of NAbs. This approach has produced novel capsids, but has the potential limitation of producing capsids with unknown transduction efficiency in humans, since these mutants are isolated from and tested in animal tissues, data from animal testing is not always transferred to humans, and there is no reliable system available to predict AAV transduction in human tissues. A third approach is to use alternative serotypes of AAV with low or no NAb cross-reactivity. Although this general 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 engineer the NAb-binding domain on the AAV capsid surface to eliminate NAb-binding sites. This strategy requires information about monoclonal antibody epitopes and the structure of the AAV virion, and is inherently limited due to the fact that NAbs derived from human serum are polyclonal, making it impossible to obtain human-derived mAbs that represent all produced NAbs. Several clinically relevant approaches have also been 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 titers of NAbs (<1:5) can inhibit AAV transduction, this strategy is only appropriate for patients with lower starting titers of AAV NAb, and requires multiple sessions of apheresis.Similarly, the use of anti-CD20 antibodies (rituximab) can achieve B cell depletion, but it takes a long time (approximately 6-9 months) and is only effective in reducing AAV NAb in a minority of subjects. The final clinical approach is to use excess empty AAV capsids as decoys for NAb. The concern remains that the addition of empty particles increases the load of AAV capsids, thereby potentially increasing the elimination of AAV-transduced cells mediated by capsid-specific cytotoxic immune responses, possibly competing with full AAV particles for effective transduction. Furthermore, empty capsids may induce greater liver inflammation than full AAV vectors. Compared with the low efficiency of NAb protection or the complexity of the above approaches, in this study a strategy with greater potential for blocking neutralizing antibody activity was demonstrated using immunoglobulin-binding protein M. With protein M, NAb evasion capacity was achieved 100-fold in vitro and over 1000-fold in vivo.

[0276] 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 antigen recognition or structural interference with the CDR regions. Protein M binds to antibodies to prevent antigen-antibody binding, but does not disrupt previously formed antigen-antibody complexes. The interaction of antibodies with 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, and thus can be incorporated into therapeutic regimens, including gene therapy previously approved by the FDA. This is advantageous 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 avoid humoral immune response-mediated clearance. Protein M also has the potential to treat autoimmune disorders caused by autoantibodies.

[0277] This study showed 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 over potential complications of high doses of protein M need to be addressed in large animals before clinical trials. This would not be a major issue if protein M is used locally for AAV administration.

[0278] In vitro studies have shown that Protein M / immunoglobulin complexes are relatively stable for long periods of time, but in vivo experiments have shown a gradual loss of Protein M function when AAV vectors are administered long after application of Protein M. It is important to understand the dynamics and kinetics of Protein M / immunoglobulin complexes in the blood, as this information will provide valuable information regarding the window for AAV infusion following administration of Protein M.

[0279] Another concern is the immunogenicity of protein M with respect to repeated administration. Since protein M is a foreign protein, it induces a humoral immune response and produces 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 portion of all Ig, so if a high dose of protein M is used for the purpose of blocking AAV NAb, the amount of protein M specific Ig will only neutralize a very small amount of protein M, and therefore the administered protein M may not affect the function of protecting 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 is capable of inducing B cell proliferation, but truncated protein M loses this function. Long-term in vivo follow-up after protein M administration should be performed.

[0280] In conclusion, the results show that protein M prevents immunoglobulins from neutralizing AAV when present at a molecular ratio of ≥2 molecules of protein M to 1 molecule of IgG. 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 for the use of protein M for protection of AAV vector virions from NAb activity in future AAV clinical trials in patients with NAbs or for rechallenge.

[0281] Example 8: Engineered Protein M variants with enhanced properties The above examples describe the use of mycoplasma protein M as an antibody blocking protein to avoid 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 features of the naturally occurring protein make it unsuitable for use as a therapeutic drug. Native protein M is not soluble but is membrane-bound by an N-terminal transmembrane domain that anchors it in the bacterial cell membrane. Furthermore, the native protein has a disordered C-terminus that can behave unexpectedly as a drug-like molecule. A native protein M truncated form (protein M TD by Grover et al., Science 343:656 (2014)), lacking the N-terminal transmembrane domain and the disordered C-terminal segment, was tested as a therapeutic antibody blocking molecule, but the key finding was that the protein has no structural stability when incubated at body temperature (37°C).

[0282] 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 of time (15 min to 1 h). Analysis using circular dichroism revealed that heating the protein to 37°C was associated with a distinct protein unfolding event within 1 h, with immediate melting of the protein occurring at 41.2°C, while continued incubation at 20°C for 2 h did not result in any unfolding events (Figure 14). Protein unfolding also correlates 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) as inputs to protein modeling, as well as engineering software called Rosetta. Free energy calculations and 3D models were used to predict which amino acid sequence changes would result in tertiary structure stabilization, outputting a list of over 850+ amino acid substitutions. A library of rationally designed variants was used to produce mutant proteins by DNA synthesis of individual and combinatorial multiple mutants. Table 4 lists 885 computationally identified point mutations that had better scores than the wild-type protein for thermostability. Table 5 lists 165 computationally identified point mutations that score substantially better (delta score above -3.0) than the wild-type protein for thermostability.

[0283] Different protein M mutants were examined using differential scanning fluoroscopy and protein melting temperatures were calculated (Figures 15, 16). The mutants were then subjected to temperature exposure at 37°C for different times 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 (18A-18C). In addition to testing analogs of protein M from M. genitalium, analogs from M. pneumonia were also produced. Some mutants were then tested by biolayer interferometry (BLI) to determine the affinity of the mutant proteins for mouse IgG, showing that the mutants can alter the affinity of the mutants for immunoglobulin substrates (Figures 21, 22). The mutants also showed increased stability as evidenced by their stability over a wider pH range than the wild-type protein M (Figure 26). MG29, one of the lead analogs that has increased stability at 37° C. and maintains nanomolar affinity for IgG, was used to test blocking of AAV neutralizing antibodies in vivo after direct immunization of mice against AAV capsid. Results of intramuscular injection of AAV formulated in phosphate buffered saline in one leg and AAV formulated with MG29 in the other leg showed that MG29 blocked AAV neutralizing antibodies in AAV-immunized mice, allowing for effective gene delivery and re-administration of AAV (FIGS. 19, 20).

[0284] The second round of rational engineering strategies using Rosetta software included altering the affinity of the mutant proteins using many human immunoglobulin structures of the average model to predict binding site modifications of the 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 generate antigenically distinct analogs that do not occur in nature. These analogs can be chimeric, mosaic, or de novo rationally altered amino acid variants 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 produced against the protein M analog. This is because the protein M analog directly competes for blocking antibody binding and antigen recognition, as well as for its own recognition by inhibitory antibodies. A saturating dose of protein M analog is in excess of the small amount of immunoglobulin produced against the protein M analog after the first immune exposure. Furthermore, the generation of Protein M analogs with increased epitopic diversity can provide an additional layer of immune evasion from Protein M inhibitory antibodies. It is contemplated that each of the listed engineering strategies can be combined or layered on top of each other to generate Protein M analogs with multiple distinct properties.

[0285] Finally, DNA codon-optimized versions of Protein M with enhanced protein product yields were produced (Figure 25). Two codon-optimized versions of the parental truncated Protein M sequence were produced: one sequence optimized for E. coli and H. sapiens codon usage, and the other optimized for H. sapiens codon usage only. The E. coli and H sapiens dual codon constructs were used to produce protein in E. coli and showed an approximately 4-fold increase in protein yield compared to the parental non-optimized plasmid. The H. sapiens only optimized constructs contain an IL-2 secretory peptide or an albumin secretory peptide sequence, allowing for secretion of the protein from mammalian cell lines for recovery of the Protein M analog from the culture medium. Protein M mutant analogs are cloned or synthesized into both types of optimized DNA sequences and used for enhanced protein production. Protein M analogs stable at 37°C can grow in human cells after secretion without being affected by protein unfolding. Additionally, glycosylation sites of Protein M analogs, such as analog MG28(N274D), have been substituted to remove glycosylation at the binding site or to add glycosylation to the outer surface. Addition of glycosylation sites to the outer surface is another mechanism to enhance immune evasion of Protein M analogs and to reduce recognition of Protein M inhibitory antibodies with multiple administrations of Protein M analogs. Finally, DNA sequences of Protein M analogs have been stably integrated into mammalian cell lines to produce secreted proteins that are stable at 37° C. and have either removed or newly added glycosylation sites.

[0286] method AAV virus production: AAV vectors were produced using a standard approach of transfecting three plasmids into HEK293 cells. Briefly, the AAV transgene plasmid pTR-CBA-Luc was co-transfected 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 by anion exchange Q-column. The purified AAV vectors were titered by qPCR using primers aimed at amplifying segments of the packaged AAV transgene.

[0287] Protein production: Plasmid pET-28b(+) encodes the protein M analog, a truncated protein from M. genitalium or M. pneumonia lacking the transmembrane domain, with 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 by Invitrogen. The pET-28b(+) plasmid was transiently transfected into BL21 / DE3 cells using an overnight starter culture. Autoinduction medium (Magic medium by 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.

[0288] Protein purification: Frozen bacterial cell culture pellets were thawed, lysed by sonication, DNAse treated, and clarified by centrifugation. The clarified bacterial lysate was dialyzed in nickel binding buffer (20 mM imidazole, 50 mM sodium phosphate, pH 7.4, 500 mM NaCl, 0.02% sodium azide) and passed over 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 over an S-100 size exclusion column, dialyzed into phosphate buffered saline with 2% glycerol, and quantified using spectrophotometry. The identity of Protein M was confirmed using SDS-PAGE protein gel electrophoresis for protein separation followed by Coomassie blue protein staining to correctly identify the Protein M bands at 45 kDa to 48 kDa.

[0289] For the production of some proteins, one of two strategies was implemented to purify protein M variants. The first protocol (Ni-Purity) was for small-scale production for NanoDSF. Lysis was performed by mixing the cell pellet 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 mixing for 30 min 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 with (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.

[0290] Western blot: After protein separation on 8% SDS-PAGE gel, proteins were transferred onto PVDF membrane. Immunoblotting was performed with anti-His primary antibody 1:1000 dilution (10 μg / ml) in 5% nonfat milk. Secondary goat anti-human IgG antibody was conjugated with horseradish peroxidase (1:10,000 dilution).

[0291] Determination of protein melting temperature and unfolding: Melting temperature (Tm) was determined using Nano Differential Scanning Fluorometry (NanoDSF) and protein unfolding was determined at different temperatures using circular dichroism assay. The inflection point of the first derivative indicates Tm or unfolding. Proteins were purified according to protocol 1 (Ni-Purity).

[0292] Solubility assay: A time course of thermal stability was performed by incubating seven aliquots of each SEC-purified protein (0.4 mg / mL) at 37° C. for different times. Precipitated proteins were pelleted by centrifuging the samples at 15,000×G for 10 min before loading onto a gel for SDS-PAGE. Proteins were purified according to protocol 2 (SEC-Purity).

[0293] Affinity assessment 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). Two-fold dilutions of each M construct ranging from 1000 nM to 15.6 nM were performed using ForteBio Kinetics buffer (PBS + detergent). Binding to anti-mouse IgG Fc Capture biosensors was assessed. Each protocol included baseline, association, and dissociation steps of 10 min, 5 min, and 5 min, 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 250-15.6 nM dilution range (n=5).

[0294] Structural modeling and rational protein engineering: Optimization of the thermal stability of analogs of protein M (PM) was achieved using an in silico rational protein engineering approach based on existing crystal structures of PM bound to immunoglobulin (4NZT and 4NZR). The Rosetta software package was used to identify mutations that stabilize the PM peptide. The first protocol involved in silico site-saturation mutagenesis to refill adjacent amino acids around the mutated residue. The second protocol involved combinatorial mutations in underfilled regions in the crystal structure. Mutants were ranked according to the difference in score between the wild-type amino acid and the substitution. Further visual inspection was performed to identify favorable mutants. Mutants were cloned and synthesized by Twist Biosciences in the pET-28b+ vector. Further design strategies were performed to alter the affinity of the binding site and generate PM analogs with distinct antigenicity.

[0295] The 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 presented in PyMol.

[0296] 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 using Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin-streptomycin.

[0297] Human IVIG for in vitro neutralization experiments: 10% 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 10After IP administration of the viral genome AAV8-FVIII, followed by a boost of the same vector 2 weeks later and a second boost 6 weeks after the first, serum was collected and pooled. Mouse serum was aliquoted and stored at -80°C for later use.

[0298] In vitro AAV neutralization assay: Nab analysis was 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 either 48- or 96-well plate formats. Human IVIG or serum was serially diluted either 2- or 10-fold. AAV-Luc was incubated with either human IVIG or mouse serum for 1 h at 4° C., followed by addition of AAV and incubation for an additional 1 h at 4° C. The AAV+serum culture was then mixed with cells suspended in serum-free medium at the time of plating. For neutralization experiments with protein M, three different incubation scenarios were tested at each step for 1 h 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 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 luciferase signal was measured in a Wallac1420 Victor2 automated plate reader. Nab titer was defined as the highest dilution at which luciferase activity was 50% lower than serum-free control.

[0299] AAV rechallenge in vivo in AAV-immunized mice: C57BL / 6 mice were immunized by IP injection of either 8E5vg or 1E9vg AAV8-GFP. Approximately one month later, serum was collected from the mice for titration by 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 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 administration, following IP administration of D-luciferin substrate.

[0300] Example 9: Kinetics of modified Protein M and fusion proteins with Protein M Biolayer interferometry was performed with serial dilutions of Protein M concentrations. Figure 22 shows exemplary data for MG29. Data from MG29 and other Protein M constructs are summarized in Tables 8, 9, and Figure 21. [Table 4] [Table 5]

[0301] Anti-human IgG Fc capture (AHC) and anti-mouse IgG Fc capture biosensors (AMC) were purchased from ForteBio. Experiments were performed at 37° C. for 10 min association and dissociation. Results were fitted to a 1:1 binding curve. The asterisk * indicates amino acid Y338. Analogs without * are N338.

[0302] Example 10: Antibody Neutralization In Vivo Serum was collected and pooled from mice serially immunized with AAV8. In vitro neutralization assays determined that the anti-AAV8 neutralization titer of the pooled serum was approximately 1:10,000. Different amounts of anti-AAV8 serum (determined by serum volume) were administered via IV injection to groups of AAV-naive mice in passive transfer experiments, followed 20–25 min later by IV injection of 2e10 viral genomes (vg) of the AAV8-luciferase reporter vector. Mice in some groups were administered protein M via IV injection 5 min prior to AAV administration. Noninvasive in vivo imaging of luciferase activity was performed 7 days later to quantify luminescence as a proxy for gene expression from the AAV8 vector, with a reduction in luminescence signal indicating AAV8 luciferase neutralization by serum. The results are shown in Figure 29. Mice given 0.0003ul of serum failed to neutralize AAV8, resulting in nearly 100% luminescent signal compared to serum-free naïve mice injected with only 2e10vg of AAV8-luciferase. Mice groups receiving increasing amounts of neutralizing serum progressively neutralized AAV8-luciferase, with approximately half of the AAV neutralized with 0.001ul to 0.003ul of serum and over 99% of the AAV neutralized with 0.3ul or more of serum. Groups of mice receiving 3ul of serum, followed by MG88 or MG118, then 2e10vg of AAV8-luciferase resulted in gene expression indicative of escape from neutralization due to suppression of neutralizing antibodies by protein M fusion proteins. The specific doses of MG88, MG29, MG29*, and MGWT* were 6.3mg, the dose of MG118 was 9mg per mouse, and the average weight of the mice was 21 grams.

[0303] Example 11: Thermal stability of disulfide mutants of Protein M Mutations were added to a stabilized version of protein M (starting) containing the following mutations: S150E, S196R, S198P, F237T, S232Q, A342V, N274D, A205P, and T355P (Table 10). Melting temperatures (Tm) were determined on the NanoDSF. Two capillaries were run on the same sample (Tm1 and Tm2). ΔTm represents the average difference between Tm1 and Tm2 compared to the starting construct. [Table 6]

[0304] Example 12: Antibody Neutralization In Vitro Figure 30 shows an in vitro neutralization assay in which high titer anti-AAV8 serum from mice was titrated in two-fold dilutions and added to wells as indicated on the X-axis. Different Protein M analogs (MG29, MG66, MG71, and MG64) were then added to the wells at the molecular ratios indicated in the figure legend and incubated for 1 hour. Saline was added to serum-only wells (filled circles). AAV8-luciferase (2x10 10 viral genome) was added to each well and incubated for 1 hour, after which 5 × 10 4 Huh7 cells were then cultured for 48 hours and the luciferase reporter signal, a surrogate for neutralization, was read and quantified.

[0305] The data show that these analogs have neutralizing antibody blocking function, thereby shifting the neutralization curve, with MG64 Fc-Protein M fusion dimer (Fc-PM) shifting the curve more than the Protein M monomer analog. Based on biolayer interferometry, MG64 has subnanomolar affinity for immunoglobulins.

[0306] The foregoing examples are illustrative of the present invention and should not be construed as limiting the present invention. 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. 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DNSAIDANKLKQAVGDIYNYRRFE RQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYP GAS IYENERASRDSEFQNEILKRAEQNGVTFDEN Sequence number 4: Modified M. genitalium protein M MG1 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNF TIPS FKAGYYDHVASDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPS F SGWSNTKATTVSTSNNLTYDKWTYFAAKGS PLY DS Y PNHT FEDVKTLAI DAKDI SALKTT I D S EKPT YL I IRGLS GNGS QLNELQL PESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKAN S FGFNPLVLGSKTNVINDL FASKPFTHIDLTQVTLQNS DNSAIDANKLKQAVGDIYNYRRFE RQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYP GAS IYENERASRDSEFQNEILKRAEQNGVTFDEN Sequence number 5: Modified M. genitalium protein M MG8 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNF TIPS FKAGYYDHVASDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPS F SGWSNTKATTVSTSNNLTYDKWTYFAAKGS PLY DQY PNHFFEDVKTLAI DAKDI SALKTT I D SEKPT YL I IRGLS GNGS QLNELQL PESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKAN S FGFNPLVLGSKTNVINDL FASKPFTHIDLTQVTLQNS DNSAIDANKLKQAVGDIYNYRRFE RQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYP GAS IYENERASRDSEFQNEILKRAEQNGVTFDEN Sequence number 6: Modified M. genitalium protein M MG13 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNF ITPSFKAGYYDHVASDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPSF SGWSNTKATTVSTSNNLTYDKWTYFAAKGS PLY DS Y PNHFFEDVKTLAI DAKDI SALKTT I D S EKPT YL I IRGLS GNGS QLNELDL PESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKAN S FGFNPLVLGSKTNVINDL FASKPFTHIDLTQVTLQNS DNSAIDANKLKQAVGDIYNYRRFE RQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYP GAS IYENERASRDSEFQNEILKRAEQNGVTFDEN Sequence number 7: Modified M. genitalium protein M MG15 SLSLNDGSYQSEIDLSSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNF TIPS FKAGYYDHVAEDGSLLSYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPRF PGWSNTKATTVSTSNNLTYDKWTYFAAKGS PLHIFFAK PLHIFF SALKTT IDS EKPT YL I IRGLS GNGS QLNELQL PESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKAN S FGFNPLVLGSKTNVINDL FASKPFTHIDLTQVTLQNS DNSAIDANKLKQAVGDIYNYRRFE RQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNHLEEAYREGDNTYRVNENYYP GAS IENERASRDEFQNEILKRAEQNGVTFDEN Figure 8: M.genitalium MG21 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNF ITPSFKAGYYDHVASDGSLLSYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPSF SGWSNTKATTVSTSNNLTYDKWTYFAAKGS Y PLHIFFAKGS DPNH SALKTT IDS EKPT YL I IRGLS GNGS QLNELQL PESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKAN S FGFNPLVLGSKTNVINDL FASKPFTHIDLTQVTLQNS DNSAIDANKLKQAVGDIYNYRRFE RQFQGGYG FYNPKYKLIV DVDDDIVERSLKELNHLEEAYREGDNI YRVNENYY P GAS IENERASRDSEFQNEILKRAEQNGVTFDEN Figure 9: M.genitalium MG22 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNF ITPSFKAGYYDHVASDGSLLSYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPSF SGWSNTKATTVSTSNNLTYDKWTYFAAKGS Y PLHIFFAKGS DPNH SALKTT IDS EKPT YL I IRGLS GNGS QLNELQL PESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKAN S FGFNPLVLGSKTNVINDL FASKPFTHIDLTQVTLQNS DNSAIDANKLKQAIGDIYNYRRFE RQFQGGN DVYNKLIKKYNK DDDLVYRSLKELNHLEEAYREGDNT YRVNENYY P GAS IENERASRDSEFQNEILKRAEQNGVTFDEN Figure 10: M.genitalium MG23 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNF TIPS FKAGYYDHVASDGSLLSYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPS F SGWSNTKATTVSTSNNLTYDKWTYDKWTYDKWTY PLHV DAKDI SALKTT ID SEKPT YL I IRGLS GNGS QLNELQL PESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKAN S FGFNPLVLGSKTNVINDL FASKPFTHIDLTQVTLQNS DNSAIDANKLKQAVGDIYRRFE RQFGLQLVYK INGLVYK DVDDIVERSLKELNHLEEAYREGDNT YRVNENYY P GAS IENERASRDSEFQNEILKRAEQNGVTFDEN Figure 11: M.genitalium MG24 SLSLNDGSYQSEIDLSSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNF TIPS FKAGYYDHVASDGSLLSYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPS F SGWSNTKATTVSTSNNLTYDKWTYDKWTYDKWTY PLHV DAKDI SALKTT ID SEKPT YL I IRGLS GNGS QLNELQL PESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKAN S FGFNPLVLGSKTNVINDL FVSKPFTHIDLTQVTLQNS DNSAIDANKLKQAVGDIYNYRRFE RQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNHLEEAYREGDNTYRVNENYYP GAS IENERASRDEFQNEILKRAEQNGVTFDEN Figure 12: M.genitalium MG27 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNF TIPS FKAGYYDHVAEDGSLLSYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPRF PGWSNTKATTVSTSNNLTYDKWTYFAAKGGS DPL DPL SALKTT ID SEKPT YL I IRGLS GNGS QLNELDL PESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKAN S FGFNPLVLGSKTNVINDL FVSKPFTHIDLTQVTLQNS DNSAIDANKLKQAIGDIYNYRRFE RQFQGYKYKYK FYKL DVDDDIVERSLKELNHLEEAYREGDNI YRVNENYY P GAS IENERASRDSEFQNEILKRAEQNGVTFDEN Figure 13: M.genitalium MG28 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNF TIPS FKAGYYDHVASDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPS F SGWSNTKATTVSTSNNLTYDKWTYFAAKGS PLY DS Y PNHFFEDVKTLAI DAKDI SALKTT I D SEKPT YL I IRGLS GDGS QLNELQL PESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKAN S FGFNPLVLGSKTNVINDL FASKPFTHIDLTQVTLQNS DNSAIDANKLKQAVGDIYNYRRFE RQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYP GAS IYENERASRDSEFQNEILKRAEQNGVTFDEN Sequence number 14: Modified M. genitalium protein M MG29 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNF TIPS FKAGYYDHVAEDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPRF PGWSNTKATTVSTSNNLTYDKWTYFAAKGS PLY DQY PNHT FEDVKTLAI DAKDI SALKTT I D SEKPT YL I IRGLS GNGS QLNELQL PESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKAN S FGFNPLVLGSKTNVINDL FVSKPFTHIDLTQVTLQNS DNSAIDANKLKQAVGDIYNYRRFE RQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYP GAS IYENERASRDSEFQNEILKRAEQNGVTFDEN Sequence number 15: Modified M. genitalium protein M MG31 SLSLNDGSYQSEIDLSSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNF TIPS FKAGYYDHVASDGSLLSYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPS F SGWSNTKATTVSTSNNLTYDKWTYDKWTYDKWTY PLHV DAKDI SALKTT ID SEKPT YL I IRGLS GNGS QLNELQL PESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKAN S FGFNPLVLGSKTNVINDL FASKPFTHIDLTQVTLQNS DNSAIDANKLKQAIGDIYNYRRFE RQFGLIK FYGLYK INGLYK DVDDDIVERSLKELNHLEEAYREGDNI YRVNENYY P GAS IENERASRDSEFQNEILKRAEQNGVTFDEN Figure 16: M.genitalium MG33 SLSLNDGSYQSEIDLSSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNF TIPS FKAGYYDHVASDGSLLSYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPS F SGWSNTKPTTVSTSNNLTYDKWTYDKWTYFEDGS PLNH DAKDI SALKTT ID SEKPT YL I IRGLS GNGS QLNELQL PESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKAN S FGFNPLVLGSKTNVINDL FASKPFTHIDLTQVTLQNS DNSAIDANKLKQAVGDIYNYRRFE RQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNHLEEAYREGDNTYRVNENYYP GAS IENERASRDEFQNEILKRAEQNGVTFDEN Figure 17: M.genitalium MG38 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNF TIPS FKAGYYDHVASDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPS F SGWSNTKATTVSTSNNLTYDKWTYFAAKGS PLY DS Y PNHFFEDVKTLAI DAKDI SALKTT I D SEKPT YL I IRGLS GNGS QLNELQL PESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKAN S FGFNPLVLGSKTNVINDL FASKPFTHIDLTQVDLQNS DNSAIDANKLKQAVGDIYNYRRFE RQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYP GAS IYENERASRDSEFQNEILKRAEQNGVTFDEN Sequence number 18: Modified M. genitalium protein M MG40 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNF TIPS FKAGYYDHVASDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPS F SGWSNTKATTVSTSNNLTYDKWTYFAAKGS PLY DS Y PNHFFEDVKTLAI DAKDI SALKTT I D SEKPT YL I IRGLS GNGS QLNELQL PESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKAN S FGFNPLVLGSKTNVINDL FASKPFTHIDLTQVPLQNS DNSAIDANKLKQAVGDIYNYRRFE RQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYP GAS IYENERASRDSEFQNEILKRAEQNGVTFDEN Sequence number 19: Modified M. genitalium protein M MG43 SLSLNDGSYQSEIDLSSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNF TIPS FKAGYYDHVAEDGSLLSYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPRF PGWSNTKATTVSTSNNLTYDKWTYFAAKGS PLHIFFAK PLHIFF SALKTT ID SEKPT YL I IRGLS GNGS QLNELQL PESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKAN S FGFNPLVLGSKTNVINDL FVSKPFTHIDLTQVTLQNS DNSAIDANKLKQAIGDIYNYRRFE RQFQGGN DVYNKLKYNKLKYK DDDLVYRSLKELNHLEEAYREGDNT YRVNENYY P GAS IENERASRDSEFQNEILKRAEQNGVTFDEN M.genitalium MG44 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNF IT PS FKAGYYDHVAEDGSLLSYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPRF PGWSNTKATTVSTSNNLTYDKWTYFAAKGGS DPL DPN SALKTT ID SEKPT YL I IRGLS GNGS QLNELQL PESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKAN S FGFNPLVLGSKTNVINDL FVSKPFTHIDLTQVTLQNS DNSAIDANKLKQAIGDIYNYRRFE RQFQGGN DVYNKLKYNKLKYK DDDLVYRSLKELNHLEEAYREGDNT YRVNENYY P GAS IENERASRDSEFQNEILKRAEQNGVTFDEN Figure 21: M.genitalium MG45 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNF IT PS FKAGYYDHVASDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPS F SGWCNTKATTVSTSNNLTYDKWTYFACKGS PLY DS Y PNHFFEDVKTLAI DAKDI SALKTT I D SEKPT YL I IRGLS GNGS QLNELQL PESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKAN S FGFNPLVLGSKTNVINDL FASKPFTHIDLTQVTLQNS DNSAIDANKLKQAVGDIYNYRRFE RQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYP GAS IYENERASRDSEFQNEILKRAEQNGVTFDEN Sequence number 22: Modified M. genitalium protein M MG46 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNF IT PS FKAGYYDHVAEDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQVPRF PGWCNTKATTVSTSNNLTYDKWTYFACKGS PLY DQY PNHT FEDVKTLAI DAKDI SALKTT I D SEKPT YL I IRGLS GNGS QLNELQL PESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKAN S FGFNPLVLGSKTNVINDL FVSKPFTHIDLTQVTLQNS DNSAIDANKLKQAVGDIYNYRRFE RQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYP GAS IYENERASRDSEFQNEILKRAEQNGVTFDEN Sequence number 23: Wild-type M. pneumoniae protein M MKLNFKIKDKKTLKRLKKGGFWALGLFGAAINAFSAVL IVNEVLRLQSGETL IASGRSGNLS FQLYSKVNQNAKSKLNS IS LT DGGYRSEI DLGDGSNFREDFRNFANNL SEAT T DAPKDLLRP VPKVEVS GL I KT SST FIT PNFKAGYYDQVAADGKTLKYYQST EY FNNRVVMP ILQTTNGTLT ANNRAYDDI FVDQGVPKFPGWFHDVDKAYYAGS NGQS EYL FKEWNYYVANGS PLYNVYPNHH FKQIKTIAFDAPRIKQGNIDGINLNLKQRNPDYVIINGLIGDGSTLKDLELPESVKKVS IN YG DYHS INVAKQIFKNVLELEFYSTNQDNNFGFNPLVLGDHTNI IN YDL BOTTLE FNY IN DLT SLEL KDNQDNI THAT CLOCK REQUIREMENTS DI Y IRRRFERQMQGYWAGGY IN DRYLVKNTNE KNVNKDNDTVYAAL KDINLHLEET YTHGGNTMYRVNENYY PGASAYEAERAT RDSE FQKEIVQRAEL IGVVFEYGV KNLRPGLKYTVKFES PQEQVALKSTDKFQPVIGSVTDMSKSVIDLIGVLRDNAEILNITNVS CHAIN ​​TVVAELKEKL DRENVFQE IN RT Document 24: Different strains of M.pneumoniae SI SLT DGGYRSEI DLGDGSNFREDFRNFANNLS EAIT DAPKDLLRPVPKVEVS GL IKT S ST F IT PNFKAGYYDQVAADGKILKYYQSTEYFNNRVVMPILQTTNGTLTANNRAYDDIFVDQGVP KFPGWFHDVDKAYYAGSNGQSEYL FKEWNYYVANGS PLYNVYPNHHFKQIKT IAFDAPRIKQ GNT DGINLNLKQRNPDYVI INGLTGDGSTLKDLELPESVKKVS I YGDYHS INVAKQI FKNVL ELEFYSTNQDNNFGFNPLVLGDHTNI I YDL FAS KP FNY I DLT SLELKDNQDNIDASKLKRAV S DI YI RRRFERQMQGYWAGGY I DRYLVKNTNEKNVNKDNDTVYAALKDINLHLEETYTHGGN TMYRVNENYY PGASAYEAE RAT RD SE FQKE IVQRAEL I GVVFE SEQ ID NO: 25: Polynucleotide encoding M. genitalium protein M, codon-optimized for both bacterial and human expression ATGGGCAGC AGC CATCAC CATCATCATCACAGC AGC GGTC TGGTGC C GC GTGGT AGCCACATGAGCCTGAGCCTGAACGATGGTAGCTACCAGAGCGAGATCGACCTG AGCGGCGGTGCCAACTTCCGTGAAAAATTCCGCAACTTTGCTAACGAGCTGAGC GAAGC CATTAC CAATAGC C CAAAGGGC C TGGATC GTC C AGTGC C GAAAAC C GAG ATCAGC GGC C TGATTAAGAC C GGTGACAACTTTATC AC C C C GAGCTTCAAGGC GG GC TACTATGATC AC GTGGC TGAGGAC GGTAGC C TGC TGAGCTACTATCAGAGCAC C GAGTAC TTTAACAAC C GTGTGCTGATGC C GATTCTGC AGAC C AC C AAC GGCAC C CTGATGGC CAACAAC C GTGGTTATGAC GAC GTGTTC C GTCAGGTGC C GC GTTTC C C GGGCTGGAGCAAC AC CAAAGC GAC CAC C GTGAGCAC C AGC AACAAC CTGAC CT AC GACAAGTGGAC CTATTTC GC C GC GAAAGGTAGC C C GCTGTAC GATCAGTATC C GAACCACACCTTTGAGGACGTGAAAACCCTGGCTATCGATGCCAAGGACATTAG C GC GCTGAAAAC C AC C ATC GATAGC GAAAAGC C GAC CTAC C TGATCATTC GC GG TCTGAGCGGCAACGGTAGCCAGCTGAACGAGCTGCAGCTGCCGGAAAGCGTGAA GAAAGTGAGCCTGTACGGCGACTATACCGGTGTGAACGTGGCCAAACAGATTTT TGCGAACGTGGTGGAGCTGGAATTCTATAGCACCAGCAAGGCGAACAGCTTCGG CTTTAACCCGCTGGTGCTGGGTAGCAAAACCAACGTGATCAACGACCTGTTCGTG AGC AAGC CGTTCAC C CACATTGAC CTGAC C CAGGTGAC C CTGCAGAACAGC GAT AAC AGC GC GATC GAC GCTAACAAGCTGAAACAGGC TGTGGGC GATATCTACAAC TATC GTC GCTTC GAGC GTCAGTTTCAGGGTTAC TTC GC C GGC GGTTACATC GATA AGTATCTGGTGAAAAAC GTGAAC AC CAACAAAGACAGC GAC GATGAC CTGGTGT AC C GCAGC CTGAAGGAACTGAAC CTGC AC C TGGAGGAAGC TTATC GTGAGGGC G AC AACAC CTACTATC GC GTGAAC GAAAACTACTATC C GGGTGC C AGCATCTAC G AGAAC GAAC GTGC GAGC C GC GATAGC GAGTTTCAGAAC GAAATTCTGAAGC GTG CGGAGCAGAACGGCGTGACCTTCGACGAAAACTAATAA SEQ ID NO: 26: Polynucleotide encoding M. genitalium protein M, codon-optimized for human expression ATGAGCCTGAGCCTGAACGATGGCAGCTACCAGAGCGAGATCGACCTGTCTGGC GGAGCCAACTTCAGAGAGAAGTTCAGAAACTTCGCCAACGAGCTGAGCGAGGCC ATCACAAACAGCCCCAAAGGCCTGGACAGACCCGTGCCTAAGACAGAGATCAGC GGCCTGATCAAGACCGGCGACAACTTCATCACCCCTAGCTTCAAGGCCGGCTACT ACGATCACGTGGCCTCTGATGGCAGCCTGCTGAGCTACTACCAGTCCACCGAGTA CTTCAACAACCGGGTGCTGATGCCCATCCTCCAGACCACCAATGGCACCCTGATG GCCAACAACAGAGGCTACGACGACGTGTTCAGACAGGTGCCCAGCTTTAGCGGC TGGTCCAATACCAAGGCCACCACCGTGTCCACCAGCAACAACCTGACCTACGAC AAGTGGACCTACTTCGCCGCCAAGGGCAGCCCTCTGTACGACAGCTACCCCAAC CACTTCTTCGAGGACGTGAAAACCCTGGCCATCGACGCCAAGGATATCAGCGCC CTGAAAACCACCATCGACAGCGAGAAGCCCACCTACCTGATCATCAGAGGACTG AGCGGCAACGGCAGCCAGCTGAATGAACTCCAGCTGCCTGAGAGCGTGAAGAAG GTGTCCCTGTACGGCGATTACACCGGCGTGAACGTGGCCAAGCAGATCTTCGCCA ATGTGGTGGAACTGGAATTCTACAGCACCAGCAAGGCCAACAGCTTCGGCTTCA ACCCTCTGGTGCTGGGCAGCAAGACCAACGTGATCAACGACCTGTTCGCCAGCA AGCCCTTCACACACATCGATCTGACCCAAGTGACCCTCCAGAACAGCGACAACA GC GCCATTGATGCCAACAAGCTGAAACAGGCCGTGGGCGACATCTACAACTACA GAAGATTCGAGCGGCAGTTCCAGGGCTACTTCGCTGGCGGCTACATCGACAAGTACCTGGTCAAGAACGTGAACACCAACAAGGACAGCGACGACGACCTGGTGTACA GAAGCCTGAAAGAGCTGAACCTGCACCTGGAAGAGGCCTACAGAGAGGGCGAC AACACCTACTACAGAGTGAACGAGAACTACTACCCAGGCGCCAGCATCTACGAG AACGAGAGAGCCAGCAGAGACAGCGAGTTCCAGAACGAGATCCTGAAGCGGGC CGAGCAGAATGGCGTGACCTTCGACGAGAACTGATGA SEQ ID NO: 27: Modified M. genitalium protein M MG47 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTG DNFITP S FKAGYYDHVAED GS LL SYYQSTEYFNNRVLMPILQTTNGTLMANNRGYD DVFRQVPRFPGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDQYPNHTFEDVKTL AIDAKDISALKTTIDSEKPTYLIIRGLSGNGSQLNELQLPESVKKVSLYGDYTGVNVA KQIFANVVELEFY S T S KAN S F GFNPLVL GS KTNVINDLFV S KPFTHIDLTQVTLQN S D NS AIDANKLKQAV GDIYNYRRFERQF QGYEAGGYIDKYLVKNVNTNKDSDDDLVY RSLKELNLHLEEAYREGDNTYYRVNENYKPGASIYENERASRDSEFQNEILKRAEQN GVTFDEN SEQ ID NO: 28: Modified M. genitalium protein M MG48 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTG DNFITP S FKAGYYDHVAED GS LL SYYQSTEYFNNRVLMPILQTTNGTLMANNRGYD DVFRQVPRFPGWCNTKPTTVSTSNNLTYDKWTYFACKGSPLYDQYPNHTFEDVKTL AIDAKDISALKTTIDSEKPTYLIIRGLSGDGSQLNELQLPESVKKVSLYGDYTGVNVA KQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFVSKPFTHIDLTQVPLQNSD NS AIDANKLKQAV GDIYNYRRFERQF QGYFAGGYIDKYLVKNVNTNKDSDDDLVY RSLKELNLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQN GVTFDEN Sequence number 29: Modified M. genitalium protein M MG49 SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTG DNFITPSFKAGYYDHVAEDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYD DVFRQVPRFPGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDQYPNHTFEDVKTL AIDAKDISALKTTIDSEKPTYLIIRGLSGNGSQLNELQLPESVKKVSLYGDYTGVNVA KQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFVSKPFTHIDLTQVTLQNSD NSAIDANKLKQAVGDIYNYRRFERQFQGYFPGGYIDKYLVKNVNTNKDSDDDLVYR SLKELNLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQNG VTFDEN Sequence number 30: Modified M. pneumoniae protein M MP29 SIS LTD GGYRS EIDL GD GSNFREDF RNFANNL SEAITDAPKDLLRPVPKVEVSGLIKTS STFITPNFKAGYYDQVAEDGKTLKYYQSTEYFNNRVVMPILQTTNGTLTANNRAYD DIFVDQGVPRFPGWFHDVDKAYYAGSNGQSEYLFKEWNYYVANGSPLYNQYPNHT FKQIKTIAFDAP RIKQ GNTD GINLNLKQRNPDYVIINGLTGD GS TLKDLELPES VKKV S IYGDYHSINVAKQIFKNVLELEFYSTNQDNNFGFNPLVLGDHTNIIYDLFVSKPFNYID LT S LELKDNQDNIDAS KLKRAV S DIYIRRRFERQMQ GYWAGGYIDRYLVKNTNEKN VNKDNDTVYAALKDINLHLEETYTHGGNTMYRVNENYYP GAS AYEAERATRD S EF QKEIVQR SEQ ID NO: 31: Modified M. genitalium protein M MG64 (secretory peptide + Fc from IgG1 + 15xGS linker + MG29dGly1): MYRMQLL SCIAL SLALVTNSAPLEPKS SDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL YITREPEVTCVVVDVSHEDPEVKFNVVYVDGVEVHNAKTKPREEQYNSTYRVVSVLT VLHQDWLNGKEYKCKV SNKALPAPIEKTI S KAKGQPREP QVYTLPP S REEMTKNQV S LTCLVKGFYP S DIAVEWE SNGQPENNYKTTPPVLD SD GS FFLY S KLTVDKS RWQ QG NVFS CSVMHEALKFHYTQKSL SL SP GAGGGS GGGS GGGS GGGMSL SLNDGSYQSEI DL SGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYY DHVAED GS LL SYYQ S TEYFNNRVLMPIL QTTD GTLMANNRGYDDVFRQVPRFP GWS NTKATTVSTSNNLYYDKWTYFAAKGSPLYDQYPNHTFEDVKTLAIDAKDISALKTTI DS EKP TYLIIRGL S GD GS QLNEL QLP ES VKKV S LYGDYTGVNVAKQIFANVVELEFY S TS CAN SF GFNPLVL GS KTNVINDLFV S KPFTHIDLTQVTL QN S DN S AIDANKLKQAV G DIYNYRRFERQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNLHLEEAYR EGDNTYYRVNENYYP GAS IYENERAS RD S EF QNEILKRAEQNGVTFDEN* * Framework 32:Specific M.genitalium antibody M MG88(Fc+15xGS antibody+MG29dGly1); MYRMQLL SCIAL SLALVTNSAPLEKS SVECPPCPAPPAAAS SVFLFPPKPKDTLMI SR TPEVTCVVVDVSAEDPEVQFNWYVDGVEVHNACTKPREEQFNSTFRVVSVLTVLHQ DWLNGKEYKCKVSNKGLPS SIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYP S DIAVEWE SNGQPENNYKTTPPMLD SD GS FF LY S KLTVDKS RWQ Q GNVF S CSVMHEALHNHYTQKSL SL SP GAGGGS GGGS GGGNDG GGMDLISSLYSLQ GANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTGDNFITPSFKAGYYDHVA ED GS LL SYYQSTEYFNNRVLMPILQTTDGTLMANNRGYDDVFRQVPRFP GWSNTKA TTVSTSNNLYDKWTYFAAKGSPLYDQYPNHTAKTKTKTKTKTKTK PTYLIIRGLSGDGSQLNELQLPESVKKVSLYGDYTGVNVAKQIFANVVELEFYSTSKA NSFGFNPLVLGSKTNVINDLFVSKPFTHIDLTQVTLQNSDNSAIDANKLKQAVGDIYN YRRFERQFQGYFAGGYIDKYLVKNVNTNKDSDDDLVYRSLKELNHLEEAYREGDN TIYRVNENYYPGASIENERASRDEFQNEILKRAEQNGVTFDEN* * Figure 33: M. genitalium MG118(A(Z-Z)+10xGS+MG29): MKFNKEQQNAFYEILHLPNLNEEQRNAFIQ SLKDDPSQSANLLAEAKKLNDAQAPG GS GGSGGSGSL SLNDGSYQ SEIDL SGGANFREKFRNFANEL SEAITNSPKGLDRPVPK TEI S GLIKTGDNFITP S FKAGYYDHVAED GS LL SYYQSTEYFNNRVLMPILQTTNGTL MANNRGYDDVFRQVPRFPGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDQYPN HTFEDVKTLAIDAKDISALKTTIDSEKPTYLIIRGL SGNGSQLNELQLPESVKKVSLYG DYTGVNVAKQIFANVVELEFY S TS KANS F GFNPLVL GS KTNVINDLFV S KP FTHIDLT QVTLQNSDNSAIDANKLKQAVGDIYNYRRFERQFQGYFAGGYIDKYLVKNVNTNKD SDDDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEI LKRAEQNGVTFDEN* SEQ ID NO: 34: Modified M. genitalium protein M MG66 (with the following mutations: S150E, S196R, S198P, F237T, S232Q, A342V, N274D, A205P, and T355P): SLSLNDGSYQSEIDLSGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEISGLIKTG DNFITPSFKAGYYDHVAEDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYD DVFRQVPRFPGWSNTKPTTVSTSNNLTYDKWTYFAAKGSPLYDQYPNHTFEDVKTL AIDAKDISALKTTIDSEKPTYLIIRGLSGDGSQLNELQLPESVKKVSLYGDYTGVNVA KQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFVSKPFTHIDLTQVPLQNSD NS AIDANKL KQ AV GDIYNYRRF ERQF QGYFAGGYIDKYLVKNVNTNKDSDDDLVY RSLKELNLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQN GVTFDEN* SEQ ID NO: 35: Modified M. genitalium protein M MG71 (MG29 with protein L B1 domain + 5xGS linker + N-terminal deletion of SLSLND): MEEVTIKANLILPGCCTQTAEFKGTFEKATSEAYAYADTLKKDNGEWTVDVADKGY TLNIKFAGGS GGS GSYQ SEIDL SGGANFREKFRNFANELSEAITNSPKGLDRPVPKTEI SGLIKTGDNFITP SF KAGYYDHVAED GS LL SYYQ STEYFNNRVLMPILQTTNGTLMA NNRGYDDVFRQVPRFPGWSNTKATTVSTSNNLTYDKWTYFAAKGSPLYDQYPNHT FEDVKTLAIDAKDISALKTTID SEKPTYLIIRGL S GNGS QLNELQLPESVKKVSLYGDY TGVNVAKQIFANVVELEFYSTSKANSFGFNPLVLGSKTNVINDLFVSKPFTHIDLTQV TLQNSDNSAIDANKLKQAVGDIYNYRRFERQF QGYFAGGYIDKYLVKNVNTNKDSD DDLVYRSLKELNLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEILK RAEQNGVTFDEN* SEQ ID NO: 36: Modified M. genitalium protein M MG86 (dimer containing GCN4 helix + 15xGS + MG66) GGCGGMKQLEDKIEELLSKIYHLENEIARLKKLIGEGGSGGGSGGGSGGGSGSGSMLSLN DGSYQSEIDLSGGANFREKFRNFANELSEAITNSKPGLDPVPKPTEISGLIKTGDNFITP SFKAGYYDHVAEDGSLLSYYQSTEYFNNRVLMPILQTTNGTLMANNRGYDDVFRQ VPRFPGWSNTKPTTVSTSNNLTYDKWTYFAAKGSPLYDQYPNHTFEDVKTLAIDAK DISALKTTIDSEKPTYLIIRGLSGDGSQLNELQLPESVKKVSLYGDYTGVNVAKQIFAN VVELEFYSTSKANSFGFNPLVLGSKTNVINDLFVSKPFTHIDLTQVPLQNSDNSAIDA NKLKQAVGDIYNYRRFERQFQGYFAGGYIDKYLVKNVNTNKDSDDLVYRSLKEL NLHLEEAYREGDNTYYRVNENYYPGASIYENERASRDSEFQNEILKRAEQNGVTFDE N*

Claims

1. A fusion protein comprising: A modified mycoplasmal protein M or a functional fragment thereof; A linker; A peptide, The modified Mycoplasma protein M or functional fragment thereof has one or more amino acid mutations that increase or maintain the pH stability or thermal stability of Mycoplasma protein M or a functional fragment thereof compared to wild-type Mycoplasma protein M or a functional fragment thereof. Fusion proteins.

2. The fusion protein of claim 1, wherein the modified Mycoplasma protein M or a functional fragment thereof has one or more mutations that enhance the affinity or avidity of the modified Mycoplasma protein M or a functional fragment thereof for an antibody.

3. The fusion protein of claim 1, wherein the peptide is a dimerization peptide (e.g., IL-GCN4, C-IL-GCN4, SEQ ID NO:36).

4. The fusion protein of claim 1 , wherein the peptide is a Fab binding polypeptide or an Fc binding polypeptide.

5. The fusion protein of claim 4 , wherein the Fab binding polypeptide is Protein L.

6. The fusion protein of claim 4, wherein the Fab binding polypeptide or the Fc binding polypeptide is Protein A or a functional fragment thereof.

7. 7. The fusion protein of claim 6, wherein the fusion protein (i) binds to the Fc portion of an immunoglobulin, (ii) reduces Fc receptor recycling of an immunoglobulin, or (iii) increases binding of the fusion protein to an immunoglobulin.

8. The fusion protein of claim 4, wherein the Fab binding polypeptide or the Fc binding polypeptide is Protein G or a functional fragment thereof.

9. 9. The fusion protein of claim 8, wherein the fusion protein (i) binds to the Fc portion of an immunoglobulin, (ii) reduces Fc receptor recycling of the immunoglobulin, or (iii) increases binding of the fusion protein to an immunoglobulin.

10. The fusion protein of claim 1 , wherein the peptide comprises a dimerization peptide and protein L.

11. The fusion protein of claim 10, comprising, starting from the N-terminus, in order: (i) the dimerization peptide, (ii) the linker, (iii) the protein L, (iv) an additional linker, and (v) the modified Mycoplasma protein M or a functional fragment thereof.

12. The fusion protein of claim 1 , wherein the peptide is an Fc-binding polypeptide.

13. The fusion protein of claim 12, wherein the Fc binding polypeptide is an Fc receptor.

14. The fusion protein of claim 1 , wherein the peptide comprises a Fab binding polypeptide and protein L.

15. The fusion protein of claim 14, comprising, in order from the N-terminus, (i) the Fab binding polypeptide, (ii) the linker, (iii) the protein L, (iv) an additional linker, and (v) the modified Mycoplasma protein M or a functional fragment thereof.

16. The fusion protein of claim 1 , wherein the peptide is an Fc domain.

17. The fusion protein of claim 16, wherein the Fc domain comprises complement binding residues for binding to an Fc receptor.

18. The fusion protein of claim 17, wherein the Fc domain has one or more mutations of complement binding residues for binding to an Fc receptor.

19. 2. The fusion protein of claim 1, wherein the peptide is an Fc receptor protein and the fusion protein (i) reduces cellular uptake of antibodies, (ii) reduces antibody recycling causing immunoglobulin depletion due to reduced antibody recycling, or (iii) increases the affinity of the fusion protein for immunoglobulins.

20. The fusion protein described in claim 1, further comprising an additional modified Mycoplasma protein M or a functional fragment thereof having one or more amino acid mutations that increase or maintain the pH stability or thermal stability of Mycoplasma protein M or a functional fragment thereof compared to wild-type Mycoplasma protein M or a functional fragment thereof.

21. The fusion protein of claim 1, The modified mycoplasmal protein M or a functional fragment thereof is derived from protein M of Mycoplasma genitalium, Mycoplasma pneumoniae, or Mycoplasma penetrans; Fusion proteins.

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