WO2018175712A1 - Recombinant adeno-associated virus-paraoxonase 1-ifii particles and the methods of making and using thereof - Google Patents

Abstract

A recombinant adeno-associated virus (AAV) that expresses a paraoxonase 1 (PON1) variant protein. A formulation comprising the recombinant AAV which can be administered to a subject and provide protection against a G-type nerve agent or an organophosphorus nerve agent if the subject is exposed to the nerve agent after having received the formulation. A method of protecting a subject, including healthy human subjects, from exposure to G-type nerve agents.

Description

RECOMBINANT ADENO-ASSOCIATED VIRUS-PARAOXONASE

1 -IFII PARTICLES AND THE METHODS OF MAKING AND USING

THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

The application claims benefit of U.S. Provisional Application 62/475,502 filed March 23, 2017, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED

RESEARCH OR DEVELOPMENT

The U.S. Government has a paid-up license in this invention and the right in limited circumstances to require the patent owner to license others on reasonable terms as provided for by the terms of Grant No. CB3945 awarded by Defense Threat Reduction Agency-Joint Science and Technology Office, Medical S&T Division, Department of the Army,

Department of Defense.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on March 19, 2018, is named 200563-0040-00-WO-572733_SL.txt and is 21,596 bytes in size.

BACKGROUND

Nerve agents are colorless, tasteless, and odorless organophosphate compounds (OP) widely used as invisible chemical weapons in modern wars and civilian societies (Jett, 2016). They are known to inhibit acetylcholinesterase (AChE) and disrupt the normal functioning of the central nervous system and incapacitate and induce seizures, and cause rapid death via respiratory paralysis (Jeyaratnam, 1990; Maxwell et al, 2006; Shih et al., 2003; Thiermann et al., 2007). Many countries are actively involved in manufacturing and stockpiling more than 12 different nerve agents that are divided into G-, GV-, V-, and T-series. The G-series of nerve agents comprises soman (GD), cyclosarin (GF), sarin (GB), and tabun (GA). As of today, there are only a few inorganic chemical therapeutics such as atropine sulfate, 2- pyridine aldoxime methyl chloride (2-PAM), midazolam, diazepam, and pyridostigmine bromide (PB) available for protection against nerve agents (Jett, 2016). However, the therapeutic potential of these antidotes of G-series nerve agents is limited; and these antidotes cannot prevent brain damage due to exposure and many other clinical symptoms, because of their inefficiency in crossing the blood brain barrier (Doctor and Saxena, 2005; Jett, 2016; Jokanovic and Prostran, 2009). Also, these non-physiological synthetic antidotes of nerve agents often come with many side-effects such as "Gulf war syndrome", a prominent condition affecting veterans with a cluster of medically unexplained chronic symptoms including dry mouth, insomnia, increased heart rate, constipation, dizziness, headache, nausea, weakness, hyperventilation, blurred vision, vomiting, diarrhea, increased lung secretions, and difficulty breathing (Kerr, 2015; Locker et al., 2017; White et al, 2016).

Therefore, identification of new therapeutic approaches with safe and broad-spectrum efficacy is essential to offer complete protection against nerve agents. Therapeutics based on bacterial proteins were found excellent alternatives to these inorganic chemical antidotes but they suffer from immunogenicity when administered to the subject and rapid clearance from the body (Brazzolotto et al., 2017; Chilukuri et al., 2005; Chilukuri et al., 2008; Reed et al., 2017). Human plasma-derived butyrylcholinesterase (BChE), a stoichiometric bioscavenger expressed in multiple organs and in plasma, has displayed a circulating half-life of about 1 1 days in humans and offered complete protection against nerve agents (Ostergaard et al., 1988; Reed et al., 2017). However, the cost to purify large quantities of BChE from human plasma has become a serious issue in using the compound as a prophylaxis against nerve agents (Lockridge et al., 2016). Due to this and several other reasons, native HuBChE and recombinant HuBChE are no longer under consideration for development as prophylactics for nerve agent toxicity. Another plasma protein, paraoxonase 1 (PONl), (Accession Number P27169.3) hydrolyzes certain pesticide compounds and GA, GB, GD, and GF in vitro but with low catalytic efficiency such that this enzyme failed to offer protection against paraoxon, GA, GB, GD, and GF toxicity in vivo (Li et al., 2000, Duysen et al., 201 1).

PON 1 -IF 1 1 is a variant characterized as having increased catalytic efficiency against

G-series nerve agents (Goldsmith et al., 2012). Although PONl -IF 1 1 protein expressed in bacteria shows high catalytic activity against nerve agents, the variant protein displays low circulation stability due to rapid clearance from the body. An adenoviral vector was created to express PON1-IF1 1 gene in mice for continuous expression of protein in the circulation. While the construct demonstrated complete protection against GD, GF, GB, and GA nerve agents, the approach failed because the vector failed to express PONl -IF 1 1 for more than a week (Mata et al., 2014; Mata, 2016). Accordingly, identifying methods of preventing damage due to nerve agent and OP exposure are needed.

BRIEF SUMMARY

Disclosed is a recombinant adeno-associated virus (AAV) virion that includes a nucleic acid sequence encoding a paraoxonase 1 (PONl) variant protein having at least about 90% sequence identity to SEQ ID NO: 6. The PON l variant has a catalytic efficiency

(kcat/K_m) of greater than 10⁶ M^"1 mm^"1 as measured in serum samples in vitro. The nucleic acid sequence encoding the PONl variant protein is operably linked to a promoter and an expression element, and upon administration of the virion to a mammal or subject, the mammal or subject produces the PON 1 -IF 1 1 variant protein.

The PONl variant protein can differ from SEQ ID NO: 6 by up to 10 amino acids (i.e., 0, 1 , 2, 3, 4, 5, 6, 7, 8, 9, and 10 amino acids). The PONl variant protein of SEQ ID NO: 6 can also differ by only having 90%) sequence identity to SEQ ID NO: 6.

The recombinant AAV virion nucleic acid sequence encodes a polypeptide sequence having about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 6. The nucleic acid sequence encodes a paraoxonase 1 -IF 1 1 (PONl -IF 1 1) variant protein of SEQ ID NO: 6 or a variant having up about 90% sequence identity or more to SEQ ID NO: 6.

The recombinant AAV virus can be AAV2, AAV8, or other AAV strain including a recombinant AAV strain.

The promoter operably linked to the PON1 variant protein can be a TBG promoter.

A formulation is provided that includes recombinant AAV virion suspended in a pharmaceutically acceptable buffer. A formulation can include about 1.0 to about 9.7x1 Oe¹³ or higher virion particles per milliliter.

A nucleic acid is provided that encodes the recombinant AAV virion. The nucleic acids encoding the recombinant AAV virion can include SEQ ID NO: 5. The nucleic acid is operably linked to the promoter in a AAV8 vector. The AAV8 vector sequence can be SEQ ID NO 1.

A method of protecting a subject against an organophosphorus (OP) compound is provided. The method may include the step of administering to a subject a recombinant AAV virion in an amount sufficient to provide up to 5x LD50 protection in the subject from exposure to the OP compound. The recombinant virion is administered in an amount of about 5xel0¹² virion particles to 10xe¹² virion particles. The OP compound can be a G-type nerve agent, such as one or more of tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), or a combination thereof. Protective levels of the PON 1 -IF1 1 variant protein for the subject using the methods and compositions described can be achieved at about 3 to about 5 days after administration of the recombinant AAV virion to the subject. The method can be for treating a human subject. In some embodiments, the virion particle can be a nucleic acid of SEQ ID NO: 1 or SEQ ID NO: 5. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. A graphic representation of adeno-associated virus (AAV8)-mediated PON1 - IF1 1 gene therapy to offer protection against nerve agents. FIG. 2. AAV8-mediated expression of PON 1 -IF 1 1 under different promoters in mice. (Panel A). AAV8 expression vectors carrying PON1 -IF1 1 under a liver specific- Thyroxine binding globulin (TBG) promoter, a muscle specific- CASI promoter composed of the cytomegalovirus immediate early promoter (CMV), chimeric chicken-P-actin (CAG), and ubiquitin C (UBC) enhancer element, and a ubiquitous cytomegalovirus promoter (CMV). (Panel B). Polyacrylamide gel electrophoresis (PAGE) analysis of the purified AAV8 particles. Recombinant AAV8 particles carrying PON1-IF1 1 were generated and then purified as mentioned in materials and methods. Before transduction, the purity of the viral particles was tested by subjecting to polyacrylamide gel electrophoresis followed by

Coomassie blue staining. A pure AAV particle is composed of a viral protein 1 (VP1), viral protein 2 (VP2), and another viral protein (VP3). (Panel C). Viral transduced mice serum displays PONl-lFl 1 activity in vitro. Mice were transduced with AAV8 particles by giving tail-vein injections. Serum was collected a week after transduction and tested for PON1 -IF1 1 activity in vitro using paraoxon pesticide as a substrate. Paraoxon pesticide is an OP compound, which is in the same class of agents as G-series nerve agents, and its hydrolysis by mice serum in vitro was monitored by measuring absorbance for 10 minutes. (Panel D). Western blot to test the expression of PON 1 -IF 1 1 in mice serum. Mice serum tested for PON1-IF 1 1 activity was further analyzed by Western blotting using PON1 antibody (Sigma- Aldrich, Catalogue # P0123). Bacterial PON1-IF1 1 protein and serum collected from mice transduced with control AAV8 were used as a positive and negative controls, respectively.

FIG. 3. Long-term expression of PON 1 -IF 1 1 under TBG promoter in viral transduced mice. (Panel A). The serum collected from mice transduced with AAV8 particles carrying PON1 -IF1 1 under three different promoters were monitored for long-term expression of PON1 -IF1 1. (Panel B). Western blot analysis of serum collected from mice transduced with AAV8 carrying PON1 -IF1 1 under TBG promoter using PON1 antibody. PON1-IF 1 1 remained intact showing its molecular size as the full-length 40 to 45 kDa species. The protein appears as a doublet due to glycosylation of the protein in the animal. FIG. 4. Viral transduced mice serum hydrolyzes G-series nerve agents efficiently in vitro. (Panel A). The Ellman assay is a qualitative colorimetric test to detect OP compounds (nerve agents) based on a modification of the enzyme acetylcholinesterase (AChE). AChE hydrolyzes acetylthiocholine (ATC), which reacts with 5,5'-dithio-Z>w 2-nitrobenzoin acid (DTNB) to produce a yellow color which is then read at 405 nm. If OP is present in a sample, it will inhibit AChE reducing color formation depending upon concentration. No absorbance at 405 nm indicates no AChE enzyme or its activity (Buffer alone). (Panel B). The Ellman assay to test hydrolysis of GD by purified bacterial PON1 -IF1 1 in vitro. A variable amount of PON1 -IF1 1 was incubated with GD at room temperature for 30 min and then a 20 μΐ of the reaction mixture was added to Ellman assay. Since the extent of hydrolysis of GD depends up on the concentration of PON 1 -IF 1 1 , color formation and absorbance at 405 nm can be used as measure of enzyme expression and activity. (Panel C). Hydrolysis of GD by AAV8 transduced mice serum. Both AAV8-control and AAV8-PON1 - IF1 1 serum were incubated with GD in vitro for 30 min and then 20 μΐ of that reaction mixture was added to the Ellman assay. (Panel D) Hydrolysis of GF by AAV8 transduced mice serum. Both AAV8-control and AAV8-PON1 -IF1 1 serum were incubated with GF in vitro for 30 min and then 20 μΐ of that reaction mixture was added to the Ellman assay. (Panel E). Hydrolysis of GB by AAV8 transduced mice serum. Both AAV8-control and AAV8-PON1-IF11 serum were incubated with GB in vitro for 30 min and then 20 μΐ of that reaction mixture was added to the Ellman assay. (Panel F). Hydrolysis of GA by AAV8 transduced mice serum. Both AAV8-control and AAV8-PON1 -IF1 1 serum were incubated with GA in vitro for 30 min and then 20 μΐ of that reaction mixture was added to the Ellman assay.

FIG. 5. AAV8-PON1-IF 1 1 transduced mice display complete long-term protection against G-type nerve agents. Mice expressing PON 1 -IF 1 1 at variable concentrations were challenged with G-type nerve agents. First, all mice were tested for complete protection against GD nerve agent giving subcutaneous injections. After 24 hours, the same mice were challenged with GF for complete protection. Similarly, mice were tested for complete protection against GB and GA after 24 hours interval. Mice were also tested for complete protection against a cocktail of all four nerve agents. Control mice expressing no PON 1 -IF 1 1 die within one or two minutes after injecting a nerve agent. All mice after being challenged with nerve agents were found to exhibit no symptoms. The same mice were tested repeatedly (total 9 times) for complete protection against nerve agents within six months. One animal expressing PONl-IFl 1 at 0.37 mg/ml in serum failed to display complete protection against 5x LD50 of GA and died as a result. One other animal was removed from the study due to loss of weight as a result of leg injury.

FIG. 6. Concentration of circulating PONl-IFl 1 protein determines the level of protection against nerve agents. We have generated a large pool of mice expressing variable amount of PONl -IFl 1 protein ranging from 0.025 mg/ml to 1.05 mg/ml serum. These animals were challenged with 2x LD50 of each nerve agent after every 24 hours to determine minimum amount of PON-IF1 1 required in the circulation to offer complete protection. Arrow indicates animals require more than that particular amount of circulating PONl -IFl 1 to survive against nerve agent. (Panel A). Circulating PONl -IFl 1 concentration-dependent protection against GD. (Panel B) Circulating PONl-IFl 1 concentration-dependent protection against GF. (Panel C) Circulating PONl-IFl 1 concentration-dependent protection against GB. (Panel D) Circulating PONl -IFl 1 concentration-dependent protection against GA. Results obtained from this study can be used to determine the minimum concentration of PONl-IFl 1 in the serum required to protect human against a particular lethal dose of a nerve agent.

FIG. 7. Enzyme linked immunoassay to test PONl -IFl 1 antibodies in viral transduced mice. We have observed tapering of PONl -IFl 1 activity after a month of viral transduction; however, PONl -IFl 1 protein levels remain unchanged (FIG. 3). The reason for tapering of PONl -IFl 1 activity could be due to antibody development and interference with enzyme activity. Enzyme linked immunoassay (ELISA) was performed using different dilutions of mice serum and bacterial PONl -IFl 1 protein as antigen. Presence of antibodies against PONl-IF l 1 are represented as relative luminescence units (RLU). FIG. 8. Intramuscular injection of AAV8-TBG-PON1-IF1 1 particles and expression of PON1 -IF1 1 in mice serum. Mice received viral particles through intravenous injections displayed a statistically significantly high level of expression of PON 1 -IF 1 1 in serum. This study was performed to test PON1-IF1 1 expression after giving viral particles through intramuscular injections. Each mouse was given 50 μΐ of saline carrying 10^{1 1} viral particles through intramuscular injections. More than six mice were used for each virus. Mice serum was collected after three weeks of injections and tested for PON1 -IF1 1 expression.

FIG. 9. A circular map of viral plasmid expression vector carrying PON1 -IF1 1 under CASI promoter. AAV8 expression vector carrying PON1 -IF1 1 under a muscle-specific CASI promoter composed of the cytomegalovirus immediate early promoter (CMV), chimeric chicken-P-actin (CAG), and ubiquitin C (UBC) enhancer element was constructed. This vector carries viral sequences- 5' inverted terminal repeat (5' ITR) and 3 ' inverted terminal repeat (3' ITR) to help in inserting the CASI-PON1 -IF1 1 cassette in the viral genome.

FIG. 10. A circular map of viral plasmid expression vector carrying PON1-IF1 1 under CMV promoter. AAV8 expression vector carrying PON 1 -IF 1 1 under a ubiquitous cytomegalovirus promoter (CMV) was constructed. This vector carries viral sequences- 5' inverted terminal repeat (5' ITR) and 3' inverted terminal repeat (3' ITR) to help in inserting the CMV-PON1 -IF1 1 cassette in the viral genome.

FIG. 11. A circular map of viral plasmid expression vector carrying PON1 -IF1 1 under TBG promoter. AAV8 expression vector carrying PON 1 -IF 1 1 under a liver specific- thyroxine binding globulin (TBG) promoter was constructed. This vector carries viral sequences- 5' inverted terminal repeat (5' ITR) and 3' inverted terminal repeat (3' ITR) to help in inserting the TBG-PON1-IF1 1 cassette in the viral genome.

FIG. 12. Amino acid sequence alignment of wild type human PON1 (SEQ ID NO: 7) with PON1 -IF1 1 (SEQ ID NO: 6). PON1-IF1 1 is a variant of the wild type human PON1 with high catalytic activity against nerve agents (Goldsmith et al., 2012). Amino acids changed are shown with boxes.

DETAILED DESCRIPTION

Here, we have developed another method to express PON1 -IF1 1 for a safe and asymptomatic long-term protection against nerve agents. Based on the results obtained from a mouse model system, just one single intramuscular shot of a therapeutic formulation given to a subject a mere three days before going to the battlefield will offer complete protection against GD, GF, GB, and GA nerve agents for at least six months. In this method, we have used an FDA-approved modified adeno-associated virus to deliver PON1-IF1 1 gene into body cells without interrupting the natural genetic code.

Nerve agents are widely used in the form of gas, vapor, and or liquid as invisible chemical weapons and pesticides in modern wars and civilian societies (Jett, 2016). These toxic chemicals are divided into G-, GV-, V-, and T-series. The G-series of nerve agents comprises soman (GD), cyclosarin (GF), sarin (GB), and tabun (GA). They inhibit AChE and disrupt the normal functioning of the central nervous system and incapacitate and induce seizures, and cause rapid death via respiratory paralysis (Jeyaratnam, 1990; Maxwell et al., 2006; Shih et al., 2003; Thiermann et al., 2007). Therefore, providing relief and remission to these clinical symptoms is necessary for protecting from toxic effects of nerve agents. Thus, the current antidotal regimen available for nerve agents includes a combination of pretreatment with a spontaneously reactivating AChE inhibitor, such as pyridostigmine bromide (PB), and post-exposure therapy with anticholinergic drugs such as atropine sulfate, oximes such as 2-PAM chloride, and anti-seizure medication such as diazepam (Gray, 1984). However, their therapeutic potential is limited to periphery and they cannot prevent brain damage and many other clinical symptoms because of their inefficiency in crossing the blood brain barrier (Doctor and Saxena, 2005; Jett, 2016; Jokanovic and Prostran, 2009). Also, these nerve agent antidotes can cause many side-effects such as "Gulf war syndrome", a prominent condition affecting veterans with a cluster of medically unexplained chronic symptoms including dry mouth, insomnia, increased heart rate, constipation, dizziness, headache, nausea, weakness, hyperventilation, blurred vision, vomiting, diarrhea, increased lung secretions, and difficulty breathing (Kerr, 2015; Locker et al., 2017; White et al., 2016). Therefore, development of new, safe, and efficient therapeutic approaches is essential to offer complete protection against nerve agents. A preferred strategy for preventing nerve agents' intoxication could be scavenging them before they reach their target, AChE in the nervous system. Protein-based therapeutics have become excellent alternatives to these inorganic chemical antidotes of nerve agents. However, immunogenicity, short circulation stability, and productions cost of proteins have become serious issues in developing protein-based therapeutics as therapeutics. Thus, gene therapy has become an excellent choice to express therapeutic proteins. But the adenovirus approach failed to express PON1 -IF1 1 for more than a week (Mata et al., 2014; Mata et al, 2016). Adenovirus vectors are also immunogenic and considered unsafe for gene therapy in healthy humans. Provided here is a new method to express PON1-IF1 1 for a safe and asymptomatic long-term protection against GD, GF, GB, and GA nerve agents. Based on the results obtained from a mouse model system, just one single intramuscular shot of a therapeutic formulation given to a subject a mere three days before going to the battlefield will offer long-term protection against GD, GF, GB, and GA nerve agents. In this method, an FDA-approved modified adeno-associated virus (AAV) is used to deliver PON1 -IF 1 1 into body cells without interrupting the natural genetic code. With the ability to deliver a transgene to both non-dividing and dividing cells, many available serotypes, and no apparent pathogenicity, AAV vectors are currently among the most frequently used viral vectors for gene therapy. More than 150 AAV-mediated drugs are being tested to cure a wide variety of diseases in clinical trials worldwide.

The novel materials and methods disclosed herein are directed to the identification of therapeutic amounts of stoichiometric and catalytic bioscavengers that can be maintained in a subject, such as a human or other mammal, in vivo for weeks and months. Newly discovered is in vivo production of the catalytic bioscavenger PON1 variant IF1 1 (PON1- IF1 1) using for example, an adeno-associated virus 8 (AAV8). The construction achieved long-term expression of the bioscavenger in vivo (up to 6 months) and in amounts that afforded protection against five LD50 doses of GD, GF, GB and GA (G-type) nerve agents.

Abbreviations and Acronyms

The following abbreviations as used herein denote the following unless indicated specifically otherwise:

AAV adeno-associated virus

AChE acetylcholinesterase

ATC acetylthiocholine

CASI Synthetic promoter contains a portion of the CMV enhancer, a portion of the chicken beta-actin promoter and a portion of the ubiquitin enhancer (Balazs et al., 201 1).

CMV cytomegalovirus

G-type G-type nerve agents

GA tabun

GB sarin

GD soman

GF cyclosarin

Hu Human

i.m. or IM intramuscular

IRES internal ribosome entry sites

i.v. or IV intravascular

i.p. or IP intraperitoneal

OP organophosphate nerve agents and organophosphorus pesticides, which include G type nerve agents

PEG polyethyleneglycol

PON1 paraoxonase 1

RLU relative luminescence units SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis

TBG thyroxine binding globulin

UBC ubiquitin C

VP viral protein, such as VP 1 , VP2, and VP3

Definitions

By "AAV virion" is meant a virion of any of the serotypes, serotypes 1-1 1 , such as

AAV8.

By "recombinant AAV virion" is a recombinant virion that expresses a transgene, wherein the transgene is a PON1 protein variant.

By "PON1 protein variant" is meant to include a PON1 variant such as IF 1 1 and others that have one or more of the following characteristics:

By "catalytic efficiency of greater than 10⁶ M^"1 min^"1" is meant the rate necessary to hydrolyze G-type nerve agent intoxication when a subject is exposed to said G-type nerve agent or OP compound. Earlier, we described the assay conditions (Mata et al., 2016).

By "operably linked" is meant that the element (e.g., transgene, promoter or other element) is present in a suitably positioned in the nucleic acid molecule and oriented for transcription to be initiated by the promoter to transcribe the transgene.

The term "control elements" refers collectively to promoter regions, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites ("IRES"), enhancers, and the like, which collectively provide for the replication, transcription and translation of a coding sequence in a recipient cell. Not all of these control elements need always be present so long as the selected coding sequence is capable of being replicated, transcribed and translated in an appropriate host cell.

The term "promoter region" is used herein in its ordinary sense to refer to a nucleotide region comprising a DNA regulatory sequence, wherein the regulatory sequence is derived from a gene that is capable of binding RNA polymerase and initiating transcription of a downstream (3 '-direction) coding sequence. By "promoter" is meant the element in the nucleic acid of the AAV that promotes transcription of the transgene, a PON1 variant. An exemplary promoter is one that targets hepatocytes (the liver), including a TBG promoter.

By "AAV expression element" would include inverted terminal repeat (ITR) elements and rep elements (i.e. the Rep inhibition sequence).

By "G-type nerve agent" is meant to include at least tabun (GA), sarin (GB), soman (GD), and cyclosarin (GF).

By "protective" is meant survival of an animal administered a construct described herein that expresses a PON1-IF1 1 protein, wherein the animal shows no signs of nerve agent intoxication when exposed to said agent or OP compound. Nerve agent intoxication signs and symptoms are listed with the CDC and include central nervous system signs (e.g., miosis, headache, restlessness, convulsions, loss of consciousness and coma), respiratory signs (e.g., rhinorrhea, bronchorrhea, wheezing, dyspnea, chest tightness, hyperpnea, and bradypnea), cardiovascular signs resulting from blood loss ( tachycardia, hypertension, bradycardia, hypotension, and arrhythmias dysrhythmias), gastrointestinal signs (abdominal pain, nausea, vomiting, diarrhea, urinary incontinence or frequency), musculoskeletal signs (weakness and/or fasciculations), and skin and mucous membrane signs (e.g., profuse sweating, lacrimation, and conjunctival injection).

By "formulation" is mean a buffered solution that is pharmaceutically acceptable in humans for administration to a human or animal of an AAV construct described herein.

By "subject" is meant to include mammals such as rodents (mice and rats), agricultural animals, canines, felines, primates, and humans.

Identifying a Paraoxonase 1 (PON1) Candidate and Vector

Since chemical antidote treatments (e.g., PB, atropine sulfate, 2-PAM chloride, and diazepam) may prevent death, but do not prevent post-exposure complications such as brain damage, one alternate approach that was under intense investigations is with the use of bioscavenger proteins, or molecules that circulate in the blood stream and rapidly scavenges OP compounds including G-type nerve agents before they reach their target AChE. There are two types of bioscavengers: stoichiometric and catalytic.

With the stoichiometric bioscavenger, one molecule of the bioscavenger neutralizes one molecule of the OP compound and by doing so the bioscavenger molecule is inactivated. Thus, a larger mass of the bioscavenger is required to neutralize a smaller mass of the OPs. Plasma-derived human (Hu) BChE (butyrylcholinesterase) is the leading candidate of this type (Broomfield et al., 1991 ; Lenz et al., 2005). Because BChE acts in a stoichiometric manner, it is estimated that a large dose of 200 mg of the pure enzyme is necessary to protect a 70 kg individual against 2x LD50 of soman (Wolfe et al., 1992). In addition, there are multiple other issues that exist with this protein for mass applications including: 1) lack of circulatory stability, recombinant Hu BChE is cleared rapidly from the blood circulation, within 30 min of protein injection (Chilukuri et al., 2005); 2) PEG-modification increased the circulatory residence time but only up to 12 hours (Chilukuri et al., 2008); 3) immunogenicity issues remained with the PEG-modified Hu BChE (Chilukuri et al., 2008), native BChE can only be purified in limited quantities as its source is outdated human plasma which is available in limited amounts; 4) solubility and administration issues exist because of the requirement of a large dose; and 5) technology is yet to be developed to enable the protein to remain in the blood circulation for weeks and months.

With catalytic bioscavengers, one molecule of the bioscavenger can hydrolyze tens, hundreds or thousands of molecules of OP molecules. Therefore, catalytic bioscavengers have inherent advantages over stoichiometric bioscavengers: 1) that they are not consumed in the process of detoxifying OP molecules; and 2) that they work at low concentrations, and therefore, significantly less protein is required for protection against multiple LDso's of OP compound (Masson, 201 1).

In the category of catalytic bioscavengers, paraoxonase 1 (PON1), a 355-amino acid and calcium-dependent glycoprotein with an estimated molecular weight of 40-45 kDa was tested as a potential candidate for use as a catalytic bioscavenger. In circulation, the enzyme exists in association with high density lipoproteins and is believed to play a leading role in drug metabolism and atherosclerosis by preventing the oxidation of low density lipoproteins (Aviram et al., 1998; Gaidukov and Tawfik, 2005; Lusis, 2000; Mackness et al., 1998;

Sorenson et al., 1999).

Two human polymorphic positions in PONl exist: Q/R at position 192 and L/M at position 55 (Draganov and La Du, 2004). The Q/R polymorphism has been shown to significantly affect the catalytic efficiency of PONl . The R192 isoform hydrolyzes paraoxon faster than the Q192 isoform, while the Q192 isoform hydrolyzes G-agents faster than the R192 isoform (Davies et al., 1996). Wild-type PONl hydrolyzes a range of OP pesticide compounds in vitro, including diazoxon, chlorpyrifos, and paraoxon. While the native PONl offered in vivo protection against diazoxon and chlorpyrifos, it failed to provide protection against paraoxon and chemical warfare nerve agents (Duysen et al., 201 1 ; Hodgins et al., 2013; Stevens et al., 2008). The failure of wild-type PONl to offer protection against paraoxon and chemical warfare nerve agents is attributed to insufficient catalytic efficiency, which results in the OP escaping the blood stream before PONl is able to reduce the level of exposure below a lethal dose (Li et al., 2000).

To enhance the catalytic efficiency of wild-type human PONl (GenBank Accession Number: P27169.3) against chemical warfare nerve agents, Goldsmith and coworkers performed DNA shuffling, high-throughput library screening, and mutagenesis to generate a number of PONl variants with increased catalytic efficiencies and the desired stereochemical preference for the more toxic P(-) isomer of each G-agent (Goldsmith et al., 2012). A number of PONl variants were identified including: G3C9, VII-D1 1, VII-D2, and I-Fl 1. We have screened these PONl variants by producing them in vitro in mammalian cells and in vivo in mouse and determined that PON1 -IF1 1 is the best candidate for protecting against G- agents (GD > GF > GB > GA ) (Mata et al., 2016) and VII-D1 1 is most active for protecting against paraoxon (Mata et al., 2014).

During the past decade, gene therapy has gained importance in the treatment of a variety of disorders including hemophilia, cystic fibrosis, patients with adenosine deaminase and ornithine transcarbamylase deficiency, and cancer (Dunbar et al., 2018). Provided here are materials and methods using a gene therapy approach that is successful to provide prophylactic protection against OP G-type nerve agents and pesticide compounds. We had previously used an adenovirus for the delivery of stoichiometric and candidate catalytic bioscavengers. Specifically, adenovirus type 5 was used, but this virus system had several draw backs when it comes to human use. The draw backs included: 1) expression of the therapeutic is transient, lasting for four to eight days; 2) causes severe side effects including cancer; and 3) a second injection of the virus will not work due to strong immune response against the virus.

As the PON1 variant IF1 1 (PON1 -IF1 1) is a catalytic bioscavenger of G-type nerve agents and expressible in mammalian cells, it was sought to determine whether an AAV vector system could be used to express a therapeutic level of a PON1 variant having the enhanced characteristics. Preferred AAV vectors include AAV8, AAV2, and AAV9, however AAV8 is preferable over AAV2 and AAV9.

Of the constructs tested, AAV8 is an exemplary vector to deliver PON 1 -IF 1 1 to animals and express protein for at least 6 months. The animals containing PON 1 -IF1 1 in their blood survived multiple exposures to lethal doses of GA, GB, GD, and GF and exhibited no nerve agent toxicity symptoms. The AAV8 / PON1 -IF1 1 construct was also shown to have low immunogenicity (low levels of anti-PONl -IFl 1 antibodies) and also low toxicity in the animals.

Constructs

AAV is a non-enveloped virus that carries 4.7-kb-long, single-stranded DNA consisting of rep and cap genes flanked by inverted terminal repeats. AAV is prevalent in humans and non-human primates and at least 13 different serotypes have been isolated to date (Gao et al., 2004; Weitzman and Linden, 201 1). Each serotype displays differential tropism, making AAV a very useful system for a specific cell or tissue type transduction and a treatment option for many diseases. All the serotypes of AAV have the ability to infect cells, transport to nucleus, uncoat, and insert its genome in the host's chromosome or remain episomal. AAVl displays high transduction efficiency of muscles, neurons, heart, and retinal pigment epithelium. AAV2 can infect many types of cancer cells, neurons, kidney, retinal pigment epithelium, and photoreceptor cells. Except AAV2, no other AAV serotype has been shown to infect the kidney. AAV4 and AAV5 serotypes infect retina and retinal pigment epithelium, respectively. While AAV6 displays strong tropism for heart, AAV7 has some bias for liver (Zincarelli et al., 2008). AAV6 is also effective in infecting airway epithelial cells (Strobel et al., 2015). AAV8 and AAV9 have displayed successful infection of lymphoma and human papilloma virus (HPV) tumors respectively (Luo et al., 2015). AAV8 is the only serotype that infects pancreas. AAV8 was extensively used to express a therapeutic gene, FIX, in the liver to treat hemophilia in clinical trials (Nathwani et al., 2014). AAV tropism was further refined by mixing the capsid proteins of one serotype with the genome of another serotype. For example, AAV2/5 serotype, that transduces neurons more efficiently than the parental AAV2, was generated by packaging AAV2 genome in AAV5 capsid proteins. Other example, the pseudotyped AAV8 and AAV9, can cross the endothelial barrier of blood vessels to target muscles (Giacca and Zacchigna, 2012). Hybrid AAV serotypes were also generated by mixing the capsid proteins of multiple serotypes with the genome of another serotype. AAV-DJ serotype that consists of a hybrid capsid is generated by mixing the capsid proteins of eight different AAV serotypes. AAV-DJ displays higher transduction efficiency than any other wild type serotype in vitro and high infectivity of a broad range of tissue in vivo. Its mutant AAV-DJ8 serotype displays high infectivity of brain. AAVHSC, a new class of genetic vector isolated from hematopoietic stem cells, has been shown to be ideal for manipulating the stem cells (Smith et al., 2014). Fifty per cent of the adult human population carries AAV neutralizing antibodies that are known for reducing the expression of a therapeutic gene (Murphy et al., 2008). Capsid engineering can greatly increase the efficiency and reduce the immunogenicity of AAV and, therefore, a wide range of mosaic or hybrid and novel vectors are generated by de novo shuffling approaches (Zinn et al., 2015). For example, the hybrid vector AAV2.5, generated by combining the muscle tropism determinants of AAVl with parental AAV2, displays immune evasion of their neutralizing antibodies (Bowles et al., 2012). By applying a similar approach, other developed hybrid vectors like AAV6.2, AAV2i8, AAVrh lO, and AAVrh32.33 were beneficial for intravenous delivery, for reduction of liver sequestration, and T-cell response in the clinic respectively (Lin et al., 2007; Mays et al., 2009; Vandenberghe et al., 2009;

Worgall et al., 2008). AAV vectors were further refined by deleting CpG motifs, known ligands of Toll-like receptor 9 (TLR9), to reduce immune response for maximal expression of a transgene in clinical trials (Faust et al., 2013). Development of cre-recombination-based AAV variants has allowed transgene expression efficiently in the central nervous system, muscle, and liver (Choudhury et al., 2016; Deverman et al., 2016). With the recent advent of AAV-CRISPR/Cas9 system for in vivo genome editing, the AAV-mediated gene therapy is poised for clinical trials to treat a wide variety of diseases and disorders (Ran et al, 2015). Thus, while AAV8 is exemplified in the examples, other AAV strains can be modified and used as described herein.

Plasmid DNA expression vectors containing the AAV inverted terminal repeat sequences, a PON1 variant gene encoding PON1 variant protein under a suitable promoter such as a TBG or CMV or CASI promoter. As depicted in FIG. 1, the plasmid DNAs are co- transfected into cells, for example in human embryonic kidney epithelial cells or HEK 293 cells. The cells are then cultured under conditions appropriate for the cell type to produce AAV particles. The culture media containing the AAV virion particles is collected and the virus particles are centrifuged as described below in the examples or as known in the art for collecting and isolating AAV virion particles for in vivo administration. The virion particles are then resuspended in a pharmaceutically acceptable buffer for use in administering to the subject animal.

The isolated virion particles are centrifuged and resuspended in a pharmaceutically acceptable buffer, such as phosphate buffered saline. Vector particles are resuspended in a range of about 10xe¹² to 10xe¹³ gene copies per milliliter (for example from about lxlOe¹³ to about 10xe¹³). The final concentration of vector particles is in the range of l .OxlOe¹³ to 9.7x10e¹³ gene copies (GC) per ml. Methods of Treatment and Formulations

The compositions described herein can be used to administer to a subject, such as a human, to provide protection against being exposed to G-type nerve agents. The subject is one at the risk of being exposed to such agents, such as certain military personnel, as well as farmers and health care personnel going in to provide aid to people and spray pesticides believed to have been exposed to G-type nerve agents. The compositions would be those to be administered to healthy human subjects.

The compositions can be administered to a subject animal intravenously and intramuscularly at a dosage of about 5xe¹³ to 10xe¹³ gene copies per milliliter wherein lxe¹³ to 3xe¹³ gene copies per kg body weight is administered. The virion particles when administered to the subject are capable of expressing a PON1 variant that produces at least about 0.05 to 4.4 mg PON1 variant protein per milliliter of bloodstream of said subject. The virion particle can also result in an animal subject expressing at least about 50 μg/ml, 75 μg/ml, 100 μg/ml, 1 0 μg/ml, 200 μg/ml, and 1 to 4.4 mg/ml of serum.

The formulations can also comprise an excipient. Excipients that can be used to protect the recombinant AAV (rAAV) virion from activity degradative conditions include, but are not limited to, detergents, proteins, e.g., ovalbumin and bovine serum albumin (BSA), amino acids (e.g., glycine), polyhydric and dihydric alcohols, such as but not limited to polyethylene glycols (PEG) of varying molecular weights, such as PEG-200, PEG-400, PEG- 600, PEG-1000, PEG-1450, PEG-3350, PEG-6000, PEG-8000 and any molecular weights in between these values, with molecular weights of 1500 to 6000 preferred, propylene glycols (PG), sugar alcohols, such as a carbohydrate (e.g., sorbitol). The detergent, when present, can be an anionic, a cationic, a zwitterionic, or a nonionic detergent. An exemplary nonionic detergent is a sorbitan ester, e.g., polyoxyethylenesorbitan monolaurate (TWEEN-20) polyoxyethylenesorbitan monopalmitate (TWEEN-40), polyoxyethylenesorbitan

monostearate (TWEEN-60), polyoxyethylenesorbitan tristearate (TWEEN-65), polyoxyethylenesorbitan monooleate (TWEEN-80), polyoxyethylenesorbitan trioleate (TWEEN-85).

The amount of the various excipients present can vary. For example, a protein excipient, such as BSA, if present, will generally be present at a concentration of between 1.0 wt. % to about 20 wt. %, preferably 10 wt. %. If an amino acid such as glycine is used in the formulations, it will generally be present at a concentration of about 1 wt. % to about 5 wt. %. A carbohydrate, such as sorbitol, if present, will be present at a concentration of about 0.1 wt. % to about 10 wt. %, between about 0.5 wt. % to about 15 wt. %, and about 1 wt. % to about 5 wt. % and any 0.5% value in between. If PEG is present, it will generally be present on the order of about 2 wt. % to about 40 wt. % or about 10 wt. % to about 25 wt. %. If propylene glycol is used in the subject formulations, it will typically be present at a concentration of about 2 wt. % to about 60 wt. % or about 5 wt. % to about 30 wt. % and any integer value in between. If a detergent such as a sorbitan ester (TWEEN) is present, it will generally be present at a concentration of about 0.05 wt. % to about 5 wt. % or about 0.1 wt. % and about 1 wt. % and every 0.05 wt. % value in between those ranges.

One exemplary aqueous virion-stabilizing formulation comprises a carbohydrate, such as sorbitol, at a concentration of between 0.1 wt. % to about 10 wt. %, preferably between about 1 wt. % to about 5 wt. %, and a detergent, such as a sorbitan ester (TWEEN) at a concentration of between about 0.05 wt. % and about 5 wt. %, preferably between about 0.1 wt. % and about 1 wt. %. Virions are generally present in the composition in an amount sufficient to provide a therapeutic effect when given in one or more doses, as defined above.

The recombinant virion containing formulations described above can be administered to the subject intramuscularly (IM), intravenously (IV), and subcutaneously.

The following examples are exemplary only and do not limit the methods and compositions to only what is exemplified in the examples. Unless otherwise indicated, all kits and equipment were used according to manufacturer's directions.

EXAMPLES The following are the materials and methods used in all the examples.

VECTOR CONSTRUCTS, AAV8 PRODUCTION AND PURIFICATION

An AAV8 expression vector constructed was tested using three different promoters: CMV, TBG, and CASI. It was expected that all the promotors would function and that we would select the promoter that would display the highest levels of PONl -IF 1 1 gene expression levels in mouse.

First, paraoxonase 1 variant IF1 1 was codon harmonized for mammalian cell expression and cloned into the pENT-CMV adenoviral transfer vector as a H/ III and Xho l fragment. A ozak sequence (CCACC) was placed in front of the initiation codon (ATG). This vector was named pENT-CMV-PONl-IFl 1.

The pENT-CMV-PONl -IFl 1 construct was used to make adenoviral particles which when injected into mice produced milligrams of PON1 -IF1 1 protein. Mice (Swiss Webster / Charles River Laboratories, Wilmington, MA) containing such levels of PONl -IF 1 1 were protected from multiple lethal doses of GA, GB, GD, and GF. The protection offered depends upon the concentration of PON1 -IF1 1 present in mouse blood, the higher the levels of PONl -IF 1 1 in mouse blood, the greater tolerance to GA, GB, GD, and GF toxicity (Mata et al., 2016).

Another PONl variant called VII-Dl 1 is more active against paraoxon toxicity in mice (Mata et al., 2014). Thus, PONl variant VII-Dl 1 is more active on paraoxon, whereas

I-Fl 1 more active against the toxicity of GA, GB, GD, and GF (Mata et al., 2014 2016).

PON 1 -IF 11 gene was taken out from pENT-CMV-PONl -IFl 1 plasmid vector and then recloned into three different AAV8 viral plasmid expression vectors to generate pAAV8-CASI-PONl -IFl l (FIG. 9), pAAV8-CMV-PONl-IFl 1 (FIG. 10), and pAAV8- TBG-PON1 -IF1 1 (FIG. 1 1) viral plasmid expression vectors. These vectors carry three different promoters to express PON1 -IF1 1 in different tissues. Since liver is the main source for the secretion of the endogenous PONl into the bloodstream, PON 1 -IF 11 was cloned under a liver specific TBG promoter (Gao et al., 2006 2006). Since intramuscular injections are relatively easy than intravenous injections, PONl-IFl 1 was cloned under a muscle specific CASI promoter (Balazs et al.). The CASI promoter contains a portion of CMV enhancer, a portion of chicken beta-actin promoter, and a portion of ubiquitin enhancer element. The CMV promoter is a widely used promoter for driving gene expression in a wide variety of mammalian cells, therefore, to have abundant expression PONl-IFl 1 was cloned under CMV promoter. The AAV8 containing CMV promoter used was a self- complimentary type (Nathwani et al., 201 1). Using this type of AAV8 containing CMV promoter, it was shown that the lag time to produce factor VIII has been significantly reduced since this process eliminated the step of making double stranded DNA formation inside the host cells. These plasmid viral expression vectors were sequenced to ensure that there were no mismatches in the PONl-IFl 1 gene sequence.

AAV8 vector particles production and characterization. Production and characterization of AAV8-TBG-PON1-IF 1 1, AA V8-CMV-PON 1 -IF 1 1 , and AAV8-CASI- PON1-IF1 1 viral vector particles were performed by different vendors on paid service. While the production and characterization of AAV8-TBG-PON 1 -IF 1 1 were performed by Vector Core Gene Therapy Program, UPENN, Philadelphia, PA, the other vendor Welgen Inc., Worcester, MA, produced and characterized both AAV8-CMV-PON1-IF1 1 and AAV8- CASI-PON1 -IF1 1 viral vector particles. Control AAV8 viral vector particles carrying no PONl-IFl 1 were also produced and characterized. These AAV8 vectors carrying PONl-IFl 1 gene sequence is flanked by the AAV inverted terminal repeats (ITRs). Thus, the AAV ITRs, consisting of only 6% of the wild type AAV8 genome, are the only AAV specific sequences packaged into the vector particles. The removal of the remaining viral genes renders the vector virus replication-defective and dependent on adenovirus helper functions provided in trans. AAV8 vectors were produced in the presence of a helper plasmid, not helper virus. AAV8-viral vectors were generated by transient transfection of HEK293 cells using three plasmids (the cis ITR-containing plasmid, the trans plasmid encoding AAV replicase and capsid genes, and the adenoviral helper plasmid), which result in the pseudotyping of vector genomes with different serotype capsid proteins. All three different AAV8 viral vector were purified by tangential flow filtration followed by iodixanol gradient purification and buffer exchange as described in (Gao et al., 2006). Routine quality control conducted for preclinical vector preparations includes determination of titer and yield by quantitative PCR and endotoxin analysis. The purified AAV8 viral vectors are resuspended in phosphate buffered saline. The final concentration of AAV8 vector particles suspended are in the range of 1 to 9.7xe¹³ gene copies (GC) per ml.

Although wild type AAV virus is dependent for replication on the presence of an adenovirus or a herpesvirus and will, in the absence of helper virus, stably integrate into the host cell genome, AAV vector genomes remain primarily episomal in target cells. AAV vectors have a low (if any) frequency of integration.

Animal experiments. Adult male mice (25-30 g body weight / Swiss Webster / Charles River Laboratories, Wilmington, MA) were housed at 20 - 26° C and were provided food and water ad libitum. Viral particles were diluted to a final concentration of lxe¹³ GC per milliliter of phosphate buffered saline and 100 μΐ of that viral suspension was given to each mouse by tail-vein injections. For intramuscular injections, 50 μΐ of the diluted viral suspension was administered to each mouse. Approximately 40 μΐ of the blood collected from each mouse at various time points post virus injection into heparin coated tubes were centrifuged at 3,000 rpm for 20 min at 4°C to collect serum and stored at -80° C till use. Serum samples were diluted in saline before measuring PON1 -IF1 1 activity in vitro using paraoxon pesticide as a substrate. The diluted serum samples were also used for SDS-PAGE gel electrophoresis, and Western blotting, in in vitro GA, GB, GD, and GF hydrolysis assays, and enzyme linked immunosorbent assay (ELISA) to measure antibody production against PON1-IF1 1. Serum samples obtained from the mouse received control vector (AAV8-TBG vector) particles were used as control in the experiments.

The experimental protocols were approved by the Animal Care and Use Committee of the US Army Medical Research Institute of Chemical Defense, Aberdeen Proving Ground,

MD and all procedures were conducted in accordance with the principles stated in the Guideor the Care and Use of Laboratory Animals (National Research Council), and the Animal Welfare Act of 1966 (P.L. 89-544), as amended. (Protocol # 1 -15-U 1046 and Title: Gene delivery using adeno-associated virus to raise blood levels of catalytic bioscavenger proteins and determination of their ability to afford protection against nerve agents in mice).

PON1 activity assay. PON1 -IF1 1 enzyme activity was determined in a 96-well format on a SpectraMax M5 (Molecular Devices, Sunnyvale, CA) series spectrophotometer according to manufacturer's directions. The enzyme assay was performed in a total 200 of 50 mM Tris-HCl assay buffer pH 7.4 carrying 10 mM CaCl₂, 2.5 mM methyl paraoxon, and 1 of serum at room temperature. The absorbance of yellow colored p-nitrophenolate formed from paraoxon [Chem Service Inc., West Chester, PA (Catalogue # N- 12816)] by enzyme hydrolysis was measured at 405 nm for 10 to 20 minutes at room temperature.

PON1 -IF1 1 levels and enzyme activity in the serum were calculated using Molar extinction coefficient (ε = 17,000 M^{" 1} cm^"1) (Mata et al., 2014) and a standard curve derived from known concentrations of paraoxon and purified bacterial PON1 -IF1 1 protein.

SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. SDS-PAGE and Western blotting were performed as described earlier (Betapudi et al., 2013). Briefly, equal volume of serum or viral suspension was mixed with 2X sample buffer (Laemmli buffer: 0.125 M Tris-HCl buffer pH 6.8, 4% SDS, 20% glycerol, 10% 2- mercaptoethanol, and 0.004% bromophenol blue) and incubated at 95° C for 2 minutes and immediately kept on ice for two minutes. The samples were mixed by vortexing for a few seconds and stored at -80° C till use. For Western blotting and Coomassie staining, 10 μΐ and 20 μΐ of the samples were loaded onto 4-15% gradient polyacrylamide gels, respectively. After electrophoresis for two hours at room temperature, proteins were transferred to nitrocellulose membrane using IBlot gel transfer apparatus (Invitrogen, CA). The membrane was rinsed in phosphate buffer saline and incubated in blocking buffer (Licor Inc., NE) for two hours at 24°C. The membrane was removed from blocking buffer and incubated in blocking buffer carrying anti-PON 1 antibody (Sigma-Aldrich, catalogue # P0123) overnight at 4°C. The membrane was removed and after washing in wash buffer (19 mM Tris-HCl pH 7.4, 137 mM NaCl, 2.7 mM KC1, and 0.01% Tween 20) for three times at room temperature, incubated in blocking buffer carrying secondary antibody conjugated with infrared dye 680 (Li-Cor Biosciences, NE) for one hour at room temperature. Protein bands were detected using Infrared Imager (Li-Cor Biosciences, NE).

In vitro hydrolysis of GD, GF, GB, and GA by mouse serum containing PON1- IF11.

Before challenging mice to nerve agents, the potential of their serum carrying PON1-IF 1 1 in hydrolyzing nerve agents in vitro was tested by performing an indirect Ellman colorimetric assay. The serum samples collected 21 days post injection of viral particles was used for in vitro agent hydrolysis assay. The hydrolysis of nerve agents was performed in a total 200 \L assay buffer at room temperature for two hours. The assay buffer consists of 100 mM MOPS pH 8.0, 10 mM CaCb, 0.5 μΜ nerve agent, and eight microliters of control or PON1 -IF 1 1 serum (1 : 10 diluted) or 0.1 μg of the purified bacterial PON1 -IF1 1 protein. Any

unhydrolyzed nerve agent in the assay buffer after incubation is detected by a micro Ellman assay as described earlier (Doctor et al., 1987). The Ellman assay is a qualitative colorimetric test to detect nerve agents based on inhibition of Acetylcholinesterase (AChE). AChE hydrolyzes acetylthiocholine (ATC) (Sigma Aldrich # A5751) which reacts with 5,5'-dithio- bis 2-nitrobenzoin acid (DTNB) (Sigma-Aldrich, D8130) to produce a yellow color which can be read at 405 nm. If nerve is present in a sample, it will inhibit AChE reducing color formation depending upon concentration. No absorbance at 405 nm indicates no AChE enzyme or its activity (Buffer alone). The micro Ellman assay was performed in 200 μΐ, of assay buffer consisting of 50 mM sodium phosphate buffer, pH 7.4, 2 mM DTNB, 10 mM ATC, one unit of AChE, and 20 μΕ of the above nerve agent hydrolysis assay mixture. The Ellman assay mixture was incubated at room temperature for 10 min and then absorbance was measured at 405 nm.

Enzyme linked immunosorbent assay (ELISA). The presence of circulating antibodies in mouse blood against PON1 -IF1 1 was determined by performing ELISA in 96 well plate. Each well was incubated with 100 μΐ, of 0.1 M sodium carbonate buffer pH 9.6 carrying purified bacterial PON1 -IF1 1 (5 μg /ml) over night at 4°C. After removing buffer, wells were rinsed with wash buffer (15 mM Tris-HCl, pH 8.0, 0.15 M NaCl, 0.05% Tween- 20 buffer) for at least three times. The remaining protein binding sites of the 96-plate wells were blocked by incubation with a 200 μΐ, solution of TBST (Tris buffered saline or TBS having polysorbate 20) containing 3% bovine serum albumin (BSA) for two hours at 24^°C. After removing blocking buffer, wells were rinsed with wash buffer and incubated with 200 of wash buffer carrying one percent BSA and mouse serum (100-2000 fold diluted) overnight at 4° C. Serum buffer was removed and washed with wash buffer for five times at room temperature. The wells were then incubated with 100 of TBST containing horseradish peroxidase-conjugated anti-mouse IgG (Sigma) and incubated for 90 min. The solution was discarded and the wells were washed with TBST for five times. The wells were then incubated with SuperSignal ELISA Pico Chemiluminescent Substrate (Thermo Fisher Scientific, catalogue # 37069) and the absorbance was measured at 425 nm in an end point mode.

Challenging animals with G-Series nerve agents. Mice expressing PON1-IF1 1 enzyme in their circulation were challenged by giving subcutaneous injections of nerve agents every day. First, 100 of saline carrying GD (1 LD₅o = 124 μg/Kg body weight) to a final concentration of 5x LD50 was given subcutaneously to each mouse and observed continuously for cholinergic symptoms (e.g., tremors) for at least an hour. Control mice expressing no PON1 -IF1 1 displayed seizers and died within one or two minutes after receiving a nerve agent. Any moribund mice were euthanized immediately. Next day, all animals survived against 5x LD50 of GD, were challenged with a 5x LD50 dose of GF (240 μg/Kg body weight) and observed continuously for cholinergic symptoms. Similarly, all survived animals against GF were challenged with a 5x LD50 dose of GB (170 μg/Kg body weight) and observed for tremors. After 24 hours, all animals survived against GB were challenged with 5x LD50 dose of GA (270 μg/kg body weight). Next day, animals survived against all four nerve agents, were again challenged with a cocktail of GD, GF, GB, and GA

(6x LD50, 1.5x LD50 each nerve agent) and observed for tremors for an hour. Thus, the same animals expressing PON 1 -IF1 1 were challenged repeatedly (at least nine time) with a 5x LD50 dose of all four nerve agents separately. Control mice were used to ensure the potency of toxicity of each nerve agent just before challenging mice expressing PON1-IF1 1.

In a separate experiment, animals were injected with different number of AAV8- TBG-PON1 -IF1 1 viral particles (lxe^9"12 GC/mouse) to express variable amount of PON1- IF1 1 enzyme in the circulation. These mice were used to determine a therapeutic concentration of the circulating PON1-IF1 1 required to offer protections against different lethal doses of nerve agents. The day before challenging the mice with nerve agents, serum from each animal was collected to determine the concentration of PON 1 -IF 1 1 in their circulation. Challenging animals was started with low doses of each nerve agent. After every 24 hours, animals were challenged with one nerve agent and observed for cholinergic symptoms. Moribund mice were euthanized immediately. Animals were challenged with different doses of nerve agents (2x to 5x LD50) and an approximate therapeutic concentration of PON 1 -IF 1 1 in the circulation required was calculated for each nerve agent.

The chemistry and hematology of mice serum expressing PON1-IF11. Animals expressing PON1-IF1 1 for more than 150 days (n=3, PON1-IF1 1 enzyme concentration ranging 1 to 1.5 mg/ml) and survived multiple challenges of 5x LD50 doses of nerve agents were euthanized and blood was collected to study hematology and serum chemistry. Blood was collected from control animals (n=3) of the same age for this study. The samples were analyzed for serum chemistry using in house Ortho Clinical Diagnostics™ VITROS™ 4600 Chemistry System. All the reagents and controls used are from the VITROS Ortho Clinical Diagnostics. Approximately, 500 μΐ, of the serum was used to study the chemistry panel. Blood samples were analyzed on an in house Sysmex XT 2000i Automated Hematology Analyzer to study hematology. All the reagents used for hematology study are from XT 2000i-Sysmex. A complete necropsy of tissues including brain, liver, heart, diaphragm, kidney, pancreas, lung, urinary bladder prostate glands, epididymides, and skeletal muscle were performed for any gross or histological changes by a board-certified pathologist. EXAMPLE 1-Testing the expression of PON1-IF11 under cell specific promoters

The mission of our research program is to establish a delivery system that is capable of maintaining a catalytic bioscavenger for weeks and months at levels affording protection against 2x to 5x LD50 doses of chemical warfare nerve agents. Earlier, we have successfully demonstrated the therapeutic potential of the catalytic bioscavenger PONl -IFl 1 against high lethal doses of GD, GF, GB, and GA nerve agents using an adenovirus-mediated mice expression system (Mata et al., 2016). However, the adenovirus expression system failed express PONl -IFl 1 in the circulation for more than a week. Here, a non-immunogenic and relatively safe the AAV8 expression system was used to deliver PONl-IFl 1 for a long-term expression in the circulation. Since liver is the main source for the secretion of the endogenous PON 1 into the circulation, PONl-IFl 1 was expressed under a liver-specific TBG promoter. The expression of PONl-IFl 1 was also tested under a muscle specific CASI promoter as well as a ubiquitous CMV promoter. Recombinant AAV8 viral particles carrying PONl -IFl 1 were generated and purified and were given to mice through tail-vein injections (FIG. 2). Three weeks later, blood was collected, serum was made and assayed for PONl-IFl 1 catalytic activity using paraoxon pesticide, an OP compound closely related to nerve agents {see Materials and Methods). As shown in FIG. 2C, paraoxon hydrolysis rate is four to five folds faster (at 10 min) in mouse blood injected with AAV8-TBG-PON1 -IF1 1 compared to the rates of paraoxon hydrolysis in mouse blood injected with AAV8-CMV- PONl-IFl 1 and AAV8-CASI-PON1 -IF1 1 or AAV8 control. Western blotting experiment reveals that the 40 to 45 kDa PONl-IFl 1 protein is readily visible in mouse serum from animals injected with AAV8-TBG-PON1-IF1 1 and it is barely or not visible at all in mice serum from animals injected with AAV control, AAV8-CMV-PON1-IF1 1 and or AAV8- CASI-PON1 -IF 1 1 (FIG. 2D). PONl -IFl 1 when expressed in vitro in mammalian cells or in vivo in mouse appears as a doublet with an approximate molecular weight of 40 to 45 kDa. This is because of the post-translational modifications, usually glycosylation, in mammalian cells. In contrast, bacterially expressed PONl -IFl 1 appears as a single band with a molecular weight of 37 kDa due to lack of posttranslational modifications. Collectively, these observations suggest that TBG promoter is ideal for the high-level expression of PONl-IF l 1 in comparison with CMV and CASI promoters. These results are somewhat surprising since earlier studies showed high level expression of HIV-neutralizing immunoglobulins under CASI promoter using AAV8 gene delivery system (Balazs et al, 201 1) and PONl-IFl 1 under CMV promoter using adenovirus expression system (Mata et al., 2016). The reason for these surprising results were not explored as high-level expression of PONl -IFl 1 in the mouse circulation was obtained under TBG promoter, the primary objective of the present study.

EXAMPLE 2 - Long-term expression of PONl-IFl 1 in mouse circulation

Since the long-term expression of a therapeutic protein in the circulation is the primary goal of the present study, mouse blood was collected at various time points post- AAV8-PON1-IF1 1 administration to study the expression of PONl -IFl 1 by performing paraoxon hydrolysis assay as described under Materials and Methods. As shown in FIG. 3A, the expression of PONl -IFl 1 occurs within three days after injection of AAV8 particle into mice. High level expression of PONl -IFl 1 (~ 2 mg/ml) was observed three weeks post injection of viral particles. After three weeks of post injection of viral particles, the expression of PONl -IF l 1 remained at a concentration of nearly 1 mg/ml levels for about six months. We have measured the expression of PONl-IFl 1 up to six months only. The long- term expression of PONl-IFl I was also confirmed by performing Western blot analysis of mouse serum (FIG. 3B). A full-length 40 to 45 kDa PONl-IFl 1 was expressed in the circulation suggesting that PONl-IFl 1 is structurally intact in mouse blood for two months. PONl -IF l 1 when expressed in vitro in mammalian cells or in vivo in mouse appear as a doublet with approximate molecular weights of 40 to 45 kDa. This is because of the posttranslational modifications, mostly glycosylation that happens in mammalian cells. In contrast, bacterially expressed PONl -IFl 1 appears as a single band with a molecular weight of 37 kDa. This is because bacteria lack the posttranslational modification machinery (Mata et al., 2014). EXAMPLE 3 - Mouse serum expressing PON1-IF11 hydrolyzes nerve agents in vitro

Before challenging the mice expressing PONl -IFl 1 in their circulation with nerve agents, their serum was tested for the hydrolysis of nerve agents in vitro by performing micro Ellman assay as described in materials and methods. The Ellman assay is a qualitative colorimetric test to detect OP compounds (e.g., nerve agents) based on a modification of the enzyme Acetylcholinesterase (AChE). AChE hydrolyzes acetylthiocholine (ATC) which reacts with 5,5'-dithio-bis 2-nitrobenzoin acid (DTNB) to produce a yellow color which is then read at 405 nm. If OP is present in a sample, it will inhibit AChE reducing color formation depending upon concentration. As shown in FIG. 4A, no absorbance at 405 nm indicates no AChE enzyme or activity (Buffer alone). The Ellman assay was performed to test hydrolysis of GD by purified bacterial PONl-IFl 1 in vitro. Variable amount of PONl - IFl 1 was incubated with GD at room temperature for 30 min and then a 20 iL of the reaction mixture was added to the Ellman assay. Since the extent of hydrolysis of GD depends up on the concentration of PONl-IFl 1 , color formation and absorbance at 405 nm can be used as measure of enzyme expression and activity. Therefore, maximum absorbance at 405 nm indicates complete hydrolysis of GD or no nerve agent (FIG. 4B). Both AAV8-control and AAV8-PON1 -IF1 1 serum were incubated with GD in vitro for 30 min and then 20 \L of that reaction mixture was added to the Ellman assay. As shown in FIG. 4C, control serum without GD developed color and showed maximum absorbance at 405 nm. However, when incubated with GD, showed minimum color development and minimum absorbance at 405 nm. But, PONl-IFl 1 expressing serum displayed formation of color development and absorbance at 405 nm steadily. Similarly, the PONl-IF l 1 expressing serum displayed steady development of color and absorbance at 405 nm suggesting hydrolysis of GF (FIG. 4D), GB (FIG. 4E), and GA (FIG. 4F).

EXAMPLE 4 -Mice expressing PONl-IFl 1 display protection against nerve agents Mice transduced with AAV8-TBG-PON1 -IF1 1 were tested for survival against nerve agents as depicted in Figure 5 and described in Materials and Methods. The rationale behind conducting this experiment was that when a control mouse expressing no PON1-IF1 1 was given a lethal dose of nerve agent (2x to 5x LD50), mouse will develop cholinergic symptoms and die within one or two minutes after injection of a nerve agent. However, if a mouse expressing PON1 -IF1 1 is given the same dose of the nerve agent, the animal is expected to display no cholinergic symptoms and survive due to hydrolysis of nerve agent in vivo. As shown in FIG. 5, PON1-IF1 1 expressing mice were challenged with all four nerve agents. Mice were challenged with one nerve agent on days indicated under post AAV8-PON1 -IF1 1 transduction, days (FIG. 5, Table 1 ). The same mice were challenged with another nerve agent as shown in Table 1 , FIG. 5. The circulating PON1-IF1 1 enzyme concentration was measured three days prior to the date of challenge (column 3 of the Table). Agent used and its concentration (column 4), tremors/symptoms if any (column 5) and percent survival (column 6) were also recorded. As shown in the Table 1 in FIG. 5, only one mouse showed tremors and died due to exposure to GB and rest of the mice survived a total of nine successive exposures, over a 40-day period. In one of the exposures (last one on day 65), GD, GB, GB, and GA were mixed at 1.5x LD50 doses as a final dose of 6x LD50. One mouse was removed from the study on day 49 (marked) due to reasons un-related to G-agent toxicity. This mouse had leg injury and having difficulties walking towards food and water (dragging with belly) and has been losing weight. Thus, 15/16 mice received complete protection against nine successive 5x LD50 doses of GD, GF, GB and GA over a 40-day period. The mouse that died due to GB challenge on day 30 had the lowest amount PON1- IF1 1 in its blood (0.37 mg/ml). Another mouse, with blood levels of PON1 -IF1 1 at 0.75 mg/ml survived 5x LD50 exposures of GB and GA. From these results, it can be concluded that 0.35 mg/ml concentration of PON1 -IF1 1 is sufficient to offer protection against 5x LD50 dose of GD and GF but is not sufficient against 5x LD50 of GB whereas 0.75 mg/ml concentration of PON 1 -IF 1 1 is more than sufficient to offer protection against 5x LD50 doses of all four G-agents. Taken together, these results suggest that PON1-IF1 1 has the ability to offer prophylactic protection against 5x LD50 doses of GD, GF, GB, and GA for weeks and months when circulating in the bloodstream at therapeutic levels.

EXAMPLE 5 -Circulating PON1-IF11 required for protecting from nerve agents From the experiments described above, it is clear that PON1-IF1 1 can offer symptom- free protection against the toxicity of GD, GF, GB, and GA and that this protection is based upon the amount of PON 1 -IF 1 1 in the mouse blood. Therefore, understanding the relationship between the circulating PON1 -IF1 1 protein concentrations and lethal doses of nerve agents is important. A large pool of mice expressing variable amount of PON1 -IF1 1 protein ranging from 0.025 mg/ml to 1.05 mg/ml serum were generated by injecting variable number of viral particles. These animals were challenged with 2x LD50 of each nerve agent after every 24 hours to determine the minimum amount of PON-IF1 1 required in the circulation to offer complete protection. Arrow indicates animals require more than that particular amount of circulating PON 1 -IF 1 1 to survive against nerve agent. (A). Circulating PON1-IF1 1 concentration-dependent protection against GD. (B) Circulating PON1-IF1 1 concentration-dependent protection against GF. (C) Circulating PON1 -IF1 1 concentration- dependent protection against GB. (D) Circulating PON1-IF1 1 concentration-dependent protection against GA. Results obtained from this study can be used to determine the minimum concentration of PON 1 -IF 1 1 in the serum required to protect human against a particular lethal dose of a nerve agent. From these studies it has been determined that the amount of PON 1 -IF 1 1 required for protection against a 2x LD50 of a nerve agent in Swiss Webster strain of mice would be: ~ 1.2 mg/KG body weight for GD, ~ 2.0 mg/Kg body weight for GF, ~ 4.8 mg/Kg body weight for GB, and ~ 9.6 mg/Kg body weight for GA, respectively. At present we are not able to translate this data for human use. Humans and mice significantly differ in their sensitivity to G-agents; LD50 doses of G-agents for mouse are significantly higher than those for humans based upon per kilogram basis. This is because of carboxylesterase which is present in mouse and not in humans. Carboxylesterase is known to hydrolyze pesticide compounds and chemical warfare G-agents.

Example 6 - AAV8-PON1-IF11 expressing mice develop antibodies against PON1-IF11

Production of antibodies to PON1 -IF1 1 were also examined in the mouse bloodstream because of the possibility the enzyme activity inhibition that may prevent the therapeutic efficacy of PON1-IF1 1 during circulation. However, as shown FIG. 7, it appears that antibody production against PON1 -IF 1 1 is quite low. Low levels of antibodies were detected at higher dilutions of the serum; such as 1 : 100. In fact, the PON1 -IF1 1 expressing animals remain protected against the lethal doses of nerve agents despite carrying antibodies at low levels and therefore, antibody production issue is not of a concern.

EXAMPLE 7 - Intramuscular injection of AAV8-TBG-PON1-IF11 particles to express

PON1-IF11 in the circulation

In all the studies described above, AAV8-TBG-PON1 -IF1 1 viral particles were given to mice intravenously, via the tail vein. Successful transduction of PON1 -IF1 1 gene and expression of protein at very high levels were consistently observed in mouse blood. One of the requirements of the Bioscavenger development program is that the medical

countermeasure/prophylactic is easily administered, such as via an IM (intramuscular) injection. Therefore, we tested if an IM injection of AAV8-TBG-PON1-IF1 1 would also transduce the expression of milligram levels PON1 -IF1 1 in mouse blood (see Materials and Methods). As shown in FIG. 8, AAV8-TBG-PON 1 -IF 1 1 injected animals but not AAV8- CMV-PON1 -IF1 1 and AAV8-CASI-PON1 -IF1 1 injected animals contained PON1-IF1 1 in their blood. On day 21, expression level of up to 1 mg/ml was noted. These data suggest an IM injection of AAV8-TBG-PON1 -IF 1 1 would also transduce therapeutic levels of PON1 - IF1 1 in mouse.

EXAMPLE 8 - Mouse hematology after the expression of PON1-IF11 for six months We established that PONl -IF l 1 is a medical counter measure/catalytic bioscavenger against GD, GF, GB, and GA toxicity. One of the criteria of the successful medical counter measure is that the counter measure itself should not be toxic to the host. This is the first time that PONl -IFl 1 has been produced in vivo in mouse for up to six months in milligram quantities. In order to test if overexpression of PONl-IFl 1 for months long has caused alterations in mouse hematology, blood samples from animals overexpressing PONl -IFl 1 for six months (n=3), animals that have survived multiple agent exposures (n=3) and control animals of same age (n=3) analyzed for comprehensive hematology as described under Materials and Methods. A few minor changes were noted in animals overexpressing PON1 - IF1 1 (Table 2). For example, percentage of monocytes was increased in the PONl-IFl 1 group relative to control and nerve agent survived groups. It has been found that monocyte count increases during chronic infections. Number of reticulocytes (percent) were increased in nerve agent treated group relative to controls and PONl -IFl 1 expressing groups. This may indicate hemolytic anemia in this group. But, overall no appreciable changes were observed between the various groups.

Table 2. Effect of viral transduction on mouse hematology

CBC Control AAV8-TBG- AAV8-TBG- PON1-IF11 PON1-IF11

(Nerve agents)

RBC (Μ/μΙ) 9.383 8.327 8.76

HGB (g/dL) 13.667 12.367 12.933

HCT (%) 41.267 38.167 40.1

MCV (fl) 43.967 46.333 45.9

MCH (Pg) 14.533 14.9 14.8

MCHC (g/dL) 33.1 32.267 32.233

RDW-CV (96) 23 22.8 25.7

RDW-SD (fl) 31.333 33.3 38.567

Ρ-.Τ (Κ/μΙ) 1965.667 1509.333 1930.667

MPV (fl) 6.833 7.033 7

PCT 1.346 1.016 1.343

P-LCR 5.633 7.2 6.566

PDW 8 8.1 8.167

RETIC (%) 5.09 5.41 11.35

RET ( /μΙ) 0.477 0.43 0.987

IRF (%) 57.633 55.533 58.9

LFR (%) 42.367 44.467 41.1

MFR (%) 16.667 17.133 15.9

HFR (%) 40.967 38.4 43

EXAMPLE 9 - The long-term expression of PON1-IF11 in mice and serum chemistry

We analyzed the serum for chemical panels and the results are shown in Table 3. Only creatinine kinase and LDH values rose significantly in the PON 1 -IF 1 1 group compared to control and nerve agent survived groups. CK levels increase during skeletal muscle injury and heart damage. Normal range for creatinine kinase in mice is 1 15-1300 U/L. This condition or observation is somewhat unexpected due to overexpression of PONl -IFl 1.

LDH levels were also elevated across all the groups. Normal LDH levels reported were 50 to 600 U/L. LDH levels indicate tissue damage but this result is also unexpected.

Collectively, based on the data in FIG. 5 and above, the AAV8-mediated catalytic scavenger gene therapy using the PONl-IFl 1 variant is safe and effective. It provides long- lasting pre-treatment strategy against G-agent toxicity in a mammalian subject.

Table 3. Effect of viral transduction on mouse serum chemistry

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US2003/0003453 to Salonen et al. (Jan. 2, 2003)

US2002/0151068 to Haley et al. (Oct. 17, 2002) AAV8 vector sequence (SEQ ID NO: 1)

ATGCCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCC GGCATTTCTGACAGCTTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCCGCCAGA TTCTGACATGGATCTGAATCTGATTGAGCAGGCACCCCTGACCGTGGCCGAGAAGCTGCA GCGCGACTTTCTGACGGAATGGCGCCGTGTGAGTAAGGCCCCGGAGGCTCTTTTCTTTGT GCAATTTGAGAAGGGAGAGAGCTACTTCCACATGCACGTGCTCGTGGAAACCACCGGGG TGAAATCCATGGTTTTGGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAA TTTACCGCGGGATCGAGCCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAAT GGCGCCGGAGGCGGGAACAAGGTGGTGGATGAGTGCTACATCCCCAATTACTTGCTCCC CAAAACCCAGCCTGAGCTCCAGTGGGCGTGGACTAATATGGAACAGTATTTAAGCGCCT GTTTGAATCTCACGGAGCGTAAACGGTTGGTGGCGCAGCATCTGACGCACGTGTCGCAG ACGCAGGAGCAGAACAAAGAGAATCAGAATCCCAATTCTGATGCGCCGGTGATCAGATC AAAAACTTCAGCCAGGTACATGGAGCTGGTCGGGTGGCTCGTGGACAAGGGGATTACCT CGGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCATACATCTCCTTCAATGCGGCCTCC AACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGCGGGAAAGATTATGAGCCTGAC TAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTTCCAGCAATC GGATTTATAAAATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTC TGGGATGGGCCACGAAAAAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCA ACTACCGGGAAGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGGTG CGTAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGTGATCTG GTGGGAGGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGA GGAAGCAAGGTGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCC CGTGATCGTCACCTCCAACACCAACATGTGCGCCGTGATTGACGGGAACTCAACGACCTT CGAACACCAGCAGCCGTTGCAAGACCGGATGTTCAAATTTGAACTCACCCGCCGTCTGG ATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAG GATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAG ACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGC AGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCAGACAGGTACCAAAACAAA TGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATG AATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCC GTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATT CATCATATCATGGGAAAGGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGAT TTGGATGACTGCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGGTTA TCTTCCAGATTGGCTCGAGGACAACCTCTCTGAGGGCATTCGCGAGTGGTGGGCGCTGAA ACCTGGAGCCCCGAAGCCCAAAGCCAACCAGCAAAAGCAGGACGACGGCCGGGGTCTG GTGCTTCCTGGCTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGGGAGCCCGTC AACGCGGCGGACGCAGCGGCCCTCGAGCACGACAAGGCCTACGACCAGCAGCTGCAGG CGGGTGACAATCCGTACCTGCGGTATAACCACGCCGACGCCGAGTTTCAGGAGCGTCTG CAAGAAGATACGTCTTTTGGGGGCAACCTCGGGCGAGCAGTCTTCCAGGCCAAGAAGCG GGTTCTCGAACCTCTCGGTCTGGTTGAGGAAGGCGCTAAGACGGCTCCTGGAAAGAAGA GACCGGTAGAGCCATCACCCCAGCGTTCTCCAGACTCCTCTACGGGCATCGGCAAGAAA GGCCAACAGCCCGCCAGAAAAAGACTCAATTTTGGTCAGACTGGCGACTCAGAGTCAGT TCCAGACCCTCAACCTCTCGGAGAACCTCCAGCAGCGCCCTCTGGTGTGGGACCTAATAC AATGGCTGCAGGCGGTGGCGCACCAATGGCAGACAATAACGAAGGCGCCGACGGAGTG GGTAGTTCCTCGGGAAATTGGCATTGCGATTCCACATGGCTGGGCGACAGAGTCATCACC ACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAGCAAATCTCC AACGGGACATCGGGAGGAGCCACCAACGACAACACCTACTTCGGCTACAGCACCCCCTG GGGGTATTTTGACTTTAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAGCGACT CATCAACAACAACTGGGGATTCCGGCCCAAGAGACTCAGCTTCAAGCTCTTCAACATCCA GGTCAAGGAGGTCACGCAGAATGAAGGCACCAAGACCATCGCCAATAACCTCACCAGCA CCATCCAGGTGTTTACGGACTCGGAGTACCAGCTGCCGTACGTTCTCGGCTCTGCCCACC AGGGCTGCCTGCCTCCGTTCCCGGCGGACGTGTTCATGATTCCCCAGTACGGCTACCTAA CACTCAACAACGGTAGTCAGGCCGTGGGACGCTCCTCCTTCTACTGCCTGGAATACTTTC CTTCGCAGATGCTGAGAACCGGCAACAACTTCCAGTTTACTTACACCTTCGAGGACGTGC CTTTCCACAGCAGCTACGCCCACAGCCAGAGCTTGGACCGGCTGATGAATCCTCTGATTG ACCAGTACCTGTACTACTTGTCTCGGACTCAAACAACAGGAGGCACGGCAAATACGCAG ACTCTGGGCTTCAGCCAAGGTGGGCCTAATACAATGGCCAATCAGGCAAAGAACTGGCT GCCAGGACCCTGTTACCGCCAACAACGCGTCTCAACGACAACCGGGCAAAACAACAATA GCAACTTTGCCTGGACTGCTGGGACCAAATACCATCTGAATGGAAGAAATTCATTGGCTA ATCCTGGCATCGCTATGGCAACACACAAAGACGACGAGGAGCGTTTTTTTCCCAGTAACG GGATCCTGATTTTTGGCAAACAAAATGCTGCCAGAGACAATGCGGATTACAGCGATGTC ATGCTCACCAGCGAGGAAGAAATCAAAACCACTAACCCTGTGGCTACAGAGGAATACGG TATCGTGGCAGATAACTTGCAGCAGCAAAACACGGCTCCTCAAATTGGAACTGTCAACA GCCAGGGGGCCTTACCCGGTATGGTCTGGCAGAACCGGGACGTGTACCTGCAGGGTCCC ATCTGGGCCAAGATTCCTCACACGGACGGCAACTTCCACCCGTCTCCGCTGATGGGCGGC TTTGGCCTGAAACATCCTCCGCCTCAGATCCTGATCAAGAACACGCCTGTACCTGCGGAT CCTCCGACCACCTTCAACCAGTCAAAGCTGAACTCTTTCATCACGCAATACAGCACCGGA CAGGTCAGCGTGGAAATTGAATGGGAGCTGCAGAAGGAAAACAGCAAGCGCTGGAACC CCGAGATCCAGTACACCTCCAACTACTACAAATCTACAAGTGTGGACTTTGCTGTTAATA CAGAAGGCGTGTACTCTGAACCCCGCCCCATTGGCACCCGTTACCTCACCCGTAATCTGT AATTGCCTGTTAATCAATAAACCGGTTGATTCGTTTCAGTTGAACTTTGGTCTCTGCGAAG GGCGAATTCGTTTAAACCTGCAGGACTAGAGGTCCTGTATTAGAGGTCACGTGAGTGTTT TGCGACATTTTGCGACACCATGTGGTCACGCTGGGTATTTAAGCCCGAGTGAGCACGCAG GGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGCAGCCGCCAAGCCGAATTCTGCAGAT ATCCATCACACTGGCGGCCGCTCGACTAGAGCGGCCGCCACCGCGGTGGAGCTCCAGCT TTTGTTCCCTTTAGTGAGGGTTAATTGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCC TGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTG TAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCC CGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGG GAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTC GGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCAC AGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGG AACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCAT CACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCA GGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGA TACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGT ATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTC AGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACG ACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGC GGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTT GGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCC GGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGC AGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGG AACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAG ATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGG TCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTT CATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCAT CTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAG CAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCC TCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGT TTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATG GCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGC AAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTG TTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGAT GCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGAC CGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAA AAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGT TGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTT CACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATA AGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTT ATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAA TAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTT GTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATC GGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGT TTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCG TCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGA GGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGG GGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCT AGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAA TGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGC GATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGG CGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGT GAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCGGGCCCCCCCTCGATCG AGGTCGACGGTATCGGGGGAGCTCGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACG CGCAGCCGCC CMV Sequence (SEQ ID NO: 2)

TCAATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTG GCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGC TCATGTCCAATATGACCGCCATGTTGGCATTGATTATTGACTAGTTATTAATAGTA ATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAA CTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGT CAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCA ATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCAT ATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATT ATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATT AGTCATCGCTATTACCATGGTGATGCGGTTTTGGC AGTACACCAATGGGCGTGG A TAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGA GTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTGCGA TCGCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTAT ATAAGCAGAGCTCGTTTAGTGAACCGTCAGATC

CASI Promoter sequence (SEQ ID NO: 3)

GGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAAC GACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAG GGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGC AGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGT AAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTT GGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCAC GTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATT TATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGCGCGCG CCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGC GGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCG GCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTG CGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCG GCTCTGACTGACCGCGTTACTAAAACAGGTAAGTCCGGCCTCCGCGCCGGGTTTT GGCGCCTCCCGCGGGCGCCCCCCTCCTCACGGCGAGCGCTGCCACGTCAGACGA AGGGCGCAGCGAGCGTCCTGATCCTTCCGCCCGGACGCTCAGGACAGCGGCCCG CTGCTCATAAGACTCGGCCTTAGAACCCCAGTATCAGCAGAAGGACATTTTAGGA CGGGACTTGGGTGACTCTAGGGCACTGGTTTTCTTTCCAGAGAGCGGAACAGGCG AGGAAAAGTAGTCCCTTCTCGGCGATTCTGCGGAGGGATCTCCGTGGGGCGGTG AACGCCGATGATGCCTCTACTAACCATGTTCATGTTTTCTTTTTTTTTCTACAGGT CCTGGGTGACGAACAG

TBG Promoter (SEQ ID NO: 4)

AGGGCTGGAAGCTACCTTTGACATCATTTCCTCTGCGAATGCATGTATAATTTCT ACAGAACCTATTAGAAAGGATCACCCAGCCTCTGCTTTTGTACAACTTTCCCTTA AAAAACTGCCAATTCCACTGCTGTTTGGCCCAATAGTGAGAACTTTTTCCTGCTG CCTCTTGGTGCTTTTGCCTATGGCCCCTATTCTGCCTGCTGAAGACACTCTTGCCA GCATGGACTTAAACCCCTCCAGCTCTGACAATCCTCTTTCTCTTTTGTTTTACATG AAGGGTCTGGCAGCCAAAGCAATCACTCAAAGTTCAAACCTTATCATTTTTTGCT TTGTTCCTCTTGGCCTTGGTTTTGTACATCAGCTTTGAAAATACCATCCCAGGGTT AATGCTGGGGTTAATTTATAACTAAGAGTGCTCTAGTTTTGCAATACAGGACATG CTAT AA A A ATGGAA AG ATGTTGCTTTCTG AGAGA

PON1-IF11 (SEQ ID NO: 5)

ATGGCCAAGCTGACCGCCCTGACACTGCTGGGACTCGGTCTGGCTCTCTTTGACG GGCAGAAGTCTTCCTTCCAGACAAGGTTCAATGTTCACAGAGAAGTCACCCCTGT TGAGCTGCCAAACTGCAATCTGGTGA A AGGGGTCGACAATGGGAGCGAGGAC AT GGAGATCCTCCCTAATGGACTGGCCTTCATTTCCTCCGGAGTGAAGTATCCAGGT ATTATGAGCTTCGACCCAGATAAGTCAGGGAAGATCCTGCTGATGGACCTGAAC GAGGAGGACCCTGTCGTTCTGGAACTCGGAATCACCGGCAACACACTCGACATA TCTTCCTTTAATCCTGCTGGCATTAGCACCTTCACAGATGAAGACAACACAGTCT ACCTGCTCGTCGTCAATAGGCCTGATTCTTCCTCAACCGTTGAGGTGTTCAAGTTT CAAGAAGAGGAGAAGTCTCTGCTGCATCTGAAAACAATCAGACATAAACTGCTG CCCAGCGTGAATGATATCGTGGCCGTTGGGCCTGAGCACTTTTATGCTACAAATG ACCATTACTTTGCTGACCCTTACCTGAAATCTTGGGAAATGCACCTCGGCCTGGC CTGGAGCTTTGTGACATACTATTCCCCAAACGATGTCAGGGTCGTTGCAGAAGGT TTCGACATGGCTAATGGAATCAATATCTCTCCAGACGGGAAGTATGTTTACATCG CCGAGCTGCTGGCCCACAAGATCCATGTTTATGAAAAGCACGCAAACTGGACCC TGACACCTCTGAAGTCCCTCGACTTCGACACACTCGTCGACAACATCTCCGTGGA CCCAGTGACCGGGGACCTGTGGGTGGGCTGTCATCCCAATGGAATGAGACTGTTC TATTACGACCCAAAGAACCCACCCGGCAGCGAGGTGCTGAGAATCCAAGATATC CTCTCTGAGGAACCTAAGGTCACCGTCGTGTATGCCGAGAACGGAACAGTTCTGC AAGGCAGCAGCGTGGCTGCAGTCTACAAAGGCAAACTCCTGATTGGAACAGTGT TCCACAAGGCTCTGTATTGCGAGCTGTAA

Claims

CLAIMS What is claimed is:

1. A recombinant adeno-associated virus (AAV) virion comprising a nucleic acid sequence encoding a paraoxonase 1 (PON1) variant protein having 90% sequence identity to SEQ ID NO: 6, wherein said PON1 variant has a catalytic efficiency (k_cat/ _m) of greater than 10⁶ M^"1 rnin^"1 as measured in serum samples in vitro, wherein said nucleic acid sequence encoding the PON1 variant protein is operably linked to a promoter and an expression element, wherein upon administration of said virion to a mammal, said mammal produces the PON1-IF1 1 variant protein.

2. The recombinant AAV virion of claim 1 , wherein the PON1 variant differs from SEQ ID NO: 6 by up to 10 amino acids.

3. The recombinant AAV virion of claim 1 , wherein said nucleic acid sequence encodes a paraoxonase 1 -IF 1 1 (PON1-IF1 1) variant protein of SEQ ID NO: 6.

4. The recombinant AAV virion of any of claims 1 to 3, wherein the AAV virus is AAV2 or AAV8.

5. The recombinant AAV virion of claim 4, wherein the AAV virus is AAV8.

6. The recombinant AAV virion of any of claims 1 to 5, wherein the promoter is a TBG promoter.

7. A formulation comprising the recombinant AAV virion of any of claims 1 to 6 suspended in a pharmaceutically acceptable buffer.

8. The formulation of claim 7, wherein the formulation comprises about 1.0 to about 9.7xl0e¹³ or higher virion particles per milliliter.

9. A nucleic acid encoding the recombinant AAV virion of any of claims 1 to 6.

10. A nucleic acid of claim 9, wherein the nucleic acid comprises SEQ ID NO: 5.

1 1. The nucleic acid of claim 9, wherein the nucleic acid operably linked to the promoter in an AAV8 vector is SEQ ID NO: 1.

12. A method of protecting a subject against an organophosphorus (OP)

compound comprising:

administering to said subject a recombinant AAV virion of any of claims 1 to 7 in an amount sufficient to provide up to 5x LD50 protection in the subject from exposure to the OP compound.

13. The method of claim 12, wherein the recombinant virion is administered in an amount of about 5xel0¹² virion particles to lOxe¹² virion particles.

14. The method of claim 12 or 13, wherein the OP compound is a G-type nerve agent selected from the group consisting of tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), or a combination thereof.

15. The method of any of claims 1 1 to 13, wherein subject protective levels of the PON1 -IF1 1 variant protein are achieved at about 3 to about 5 days after administration of said recombinant AAV virion to the subject.

16. The method of any of claims 12 to 15, wherein said subject is a human.

17. The method of any of claims 12 to 16, wherein the virion particle comprises a nucleic acid of SEQ ID NO: 1 or SEQ ID NO: 5.