Non Technical Summary
The danger of gene doping presents a rising threat to the performance horse industry for which regulatory bodies have appointed subcommittees to determine how it will be controlled.While racing and performanceauthorities list prohibited substances, as testing sensitivity improves, doping strategies change.Novel techniquessuch as gene manipulationare not detectable using current anti-doping methods. As a result, anti-doping programs must become more robust and incorporate additional testing modalities to protect the welfare and wellbeing of performance horse athletes. Therefore, the goal of the work is to develop a low-cost, stall-side test for rapid detection of gene doping vectors and to determine the relative time course of detecting a commonly used doping vector. The research will be the first to determine a time course of vector detection in horses, and will result in the development of arobust, sensitive, and user-friendly test. The ability to detect gene doping with high sensitivity and understanding the timeline for proper detection will aid in discouraging gene doping, conserve the welfare and integrity of performance horses, and provide the foundation for which new policies and regulations are based.

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
315	3810	1040	100%

Knowledge Area
315 - Animal Welfare/Well-Being and Protection;

Subject Of Investigation
3810 - Horses, ponies, and mules;

Field Of Science
1040 - Molecular biology;

Goals / Objectives
Thelong-termgoalis to protect the health and welfare of performance and working horses. In the near term, to evaluate the duration of detection of a common viral vector for gene delivery using established techniques that will validate the development of anovel, more sensitivetesting method that may be used stall-side to improve detection methods.Objectives/Specific AimsAim 1:To develop a low-cost, stall-side test using the CRISPR/Cas system for the rapid detection of gene doping vectors.Aim 2:To determine the time course for detection of rAAV2 DNA sequences and expressed gene in plasma, skeletal muscle, and synovial fluid following intramuscular (IM) and intra-articular (IA) administration comparing CRISPR/Cas with ddPCR and NGS for validation.

Project Methods
Aim 1Viral Vector ProductionA recombinant AAV serotype 2 (AAV2) will be generated by Charles River (Rockville, MD) employing a cGMP production of clinical grade viral particles. Many other rAAV serotypes use the core inverted terminal repeats (ITR) from rAAV2, making them a valuable target for many gene doping vectors and are known to effectively transduce both muscle and cartilage. The rAAV will consist of a green fluorescent protein (GFP) transgene flanked by AAV2 ITRs and under control of the CAG promoter. The components are packaged into the AAV capsid and are the only remaining elements of the rAAV after transduction of cells. Here, the GFP is used as a fluorescent marker when expressed in the target cells. CAG-GFP represents aproof-of-conceptfor any potential promoter and gene combination, as the molecular techniques for detection of these sequences is thesame for any gene or promoter sequence used for doping.CRISPR/Cas Detection SystemWe will optimize and adapt the SHERLOCK v2 system for detection of rAAV2 viral genome targets, the promoter. and the transgene delivered by the rAAV2 vector (CAG-GFP). This detection system relies on steps outlined by Gootenberg et al. (2017). The first step is isothermic amplification of the nucleic acid target followed by transcription of amplified DNA to produce RNA. Once bound, Cas13a activates and cleaves the reporter creating a fluorescent signal. The system can be divided into two phases or combined into a single detection system. Specificity of the SHERLOCK system comes from design of crRNA to precise target sequences while adhering to guidelines for the protospacer flanking site (PFS) which can alter enzymatic activity. A minimum of two crRNAs will be designed to each target sequence: rAAV2 inverted terminal repeats, CAG promoter, and GFP coding region using CRISPR-RT software. Optimization will be performed using serial dilution of proteinase K and treated AAV2 stocks and compared to NGS approaches to determine sensitivity and the determination of the fluorescent threshold for a positive reaction.Nucleic acid amplification and T7 promoter incorporation will be performed using isothermal recombinase polymerase amplification (RPA) for DNA or combined with reverse transcriptase for RNA (RT-RPA; TwistDX). Subsequently, T7 transcription and target detection will be performed via Cas13a-crRNA complexes. Fluorescence will be measured using a Synergy H1 microplate reader (Biotek Instruments, Inc.). Fluorescent intensity at 5, 15, and 30 minutes will be compared to detection of similar nucleic acid targets using NGS. Positive controls will be derived from serial dilution of the rAAV2-CAG-GFP stock.Aim 2: Fourteen healthy 1- to 3-year-old horses of similar breeding will be used in a completely randomized design for a 112-day study. Horses will be stratified to treatments by initial body weight, age, and sex, and be fed to meet or slightly exceed 100% NRC requirements. Horses will be group housed withad libitumaccess to grass forage and water, and will receive normal preventative health care. Prior to enrolling in the study, a blood sample will be obtained via jugular venipuncture to test for the presence of existing AAV2 antibodies. Additionally, a complete blood cell count and blood chemistry will be performed by Texas A&M University Diagnostic Laboratory (College Station, TX). Horses meeting the inclusion criteria will receive 1 of 3 treatments based on route of administration and presence or absence of vector genomes. Treatments will consist of iso-volumetric injections of 0 vector genomes (sterile phosphate buffered saline only, CON;n= 2) or injections of 1x1012vector genomes IM (TRT-IM;n= 6) or IA (TRT-IA;n= 6) in 3 mL sterile phosphate buffered saline. Blood samples will be obtained via an intravenous jugular catheter prior to injection (h 0) and after injection at 0.5, 1, 2, 3, 6, 8, 12, 24, 27, 31, and 48 hours. Additional samples will be obtained by jugular venipuncture at d 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 14, 21, 28, 35, 56, 84, and 112 d post-administration. Blood will be collected into K2EDTA vacutainers and stored at -80°C. Carpal arthrocentesis will be performed by a single collaborator to ensure procedural uniformity. Injections (CON or TRT-IA) will be administered medial to the extensor carpi radialis tendon in the palpable depression between the radial and third carpal bones to a depth of approximately 12 mm. Synovial fluid will be collected via sterile arthrocentesis prior to injection (h 0) and on days 7, 28, 56, 84, and 112 post-injections. Synovial fluid will be centrifuged at 3000 xg for 5 minutes with the supernatant aliquoted to 1.2 mL polypropylene tubes for storage at -80°C. To determine AAV2 transduction efficiency, muscle biopsy of the semitendinosus will be obtained at baseline (h 0) and on days 7, 28, and 56 post-administrations. Baseline biopsy will be performed at a site ventral to the anticipated treatment injection site to avoid interference with the treatment site. Injections (CON and TRT-IM) will be administered intramuscularly to the semitendinosus muscle dorsal to the initial biopsy site. Subsequent biopsies of the semitendinosus muscle will be performed within 1 cm of the rAAV-CAG-GFP injection site marked by suture. The muscle biopsy will be collected using a 14-gauge semi-automatic biopsy instrument at a standard depth of 5 cm. Muscle samples will be divided, and a portion snap frozen in liquid nitrogen for tissue transduction confirmation via NGS and ddPCR or immediately placed on an inverted microscope with epifluorescence to confirm GFP expression, indicating functional rAAV-CAG-GFP. Plasma samples will be subjected to ddPCR following guidelines described by Marr and Prantner (2020) for determination of the presence of rAAV2. Briefly, nucleic acids will be extracted from samples using the MagMax Cell-Free Total Nucleic Acid Isolation Kit (ThermoFisher Scientific). Primers and probe(s) will be designed using an AAV2 reference genome. Primers will be used in ddPCR reactions with QX200 ddPCR EvaGreen Supermix (Bio-Rad) as previously described by Dobnik et al., (2019). Plates will then be transferred to the C1000 Touch Thermal Cycler (Bio-Rad) and data analysis will be performed using the QuantaSoft software (Bio-Rad). For this project, NGS will be performed by Washington University Genome Engineering and iPSC Center (GEIC). Briefly, 10mL extracted nucleic acid from each plasma sample will be aliquoted to a 96-well plate and submitted to GEIC with the three target sequences to be detected. The NGS will be targeted on the AAV2 inverted terminal repeat region, the CAG promoter, and the GFP gene. GEIC will design pooled primer sets, prepare the cDNA library, and perform deep sequencing using MiSeq system (Illumina) following standardized GEIC protocols. Automated cluster generation will be performed in MiSeq and sequencing read counts will be processed as fastq files and returned in CSV and TXT file formats. Read counts will be summarized and compared to serial dilutions of spiked rAAV2-CAG-GFP in plasma and non-treated control plasma which will serve as controls to determine detection limit. Following optimization of the SHERLOCK system inaim 1, the most effective crRNAs will be identified and the best two used to test plasma samples. Cell-free DNA will be extracted from 2 mL frozen equine plasma or synovial fluid using methods described inaim 1.Positive controls will be derived from serial dilution of rAAV2-CAG-GFP stock in control equine plasma.Statistical AnalysisAll data will be analyzed using PROC MIXED in SAS v9.4 (SAS Inst., Inc.). Horse will serve as a random effect with main effects of treatment, time, and treatment´time. The model will include an autoregressive repeated statement for time. Significance will be determined whenP≤0.05 and a trend when 0.05 ≤P≤ 0.10.