Contents of Kit
1. AAV6 Capsid Coated Microtiter Plate, 12 × 8 wells
2. Negative Control, lyophilized powder
3. Anti-AAV6 Positive Control, lyophilized powder
4. Cut-off Control, lyophilized powder
5. Anti-human IgG Conjugate, ready to use, 1 × 12 ml
6. Sample Diluent, ready to use, 1 × 50 ml
7. Wash Buffer, 20 × concentrate, 1 × 50 ml
8. TMB Substrate, ready to use, 2 × 6 ml
9. Stop Solution, ready to use, 1 × 7 ml
10. Instruction Manual
Storage
1. All reagents should be stored at 2°C to 8°C for stability.
2. All the reagents and wash solutions should be used within 12 months from manufacturing date.
3. Before using, bring all components to room temperature (18-25°C). Upon assay completion ensure all components of the kit are returned to appropriate storage conditions.
4. The Substrate is light-sensitive and should be protected from direct sunlight or UV sources.
General Description
Researchers have used AAV-based vectors in pre-clinical research and in clinical trials in which AAV-based vectors have demonstrated a good safety profile. AAVs have also demonstrated lasting therapeutic gene expression following a single treatment in preclinical and clinical studies.
One of the major challenges in AAV-based gene therapy is the presence of circulating anti-AAV neutralizing antibodies, which can pre-exist in patients and may prevent successful gene transfer. High levels of circulating anti-AAV neutralizing antibodies can develop after a single administration of gene therapy and can prevent successful gene transfer in patients.
Modeling Adeno-Associated Viral Vector 6-mediated In Vivo Gene Delivery to Expanded Non-Mobilized Haemopoietic Stem Cells from Transfusion-dependent Thalassemia Patients in a Humanized Mouse
Janani Ramesh, Karthikeyan Kandasamy, Nur Nazneen Yusof, Ihsan Sujuandy, Choong Tat Keng, Liwei Chen, Bing Shao Chia, Min Liu, Zhisheng Her, Marek Kukumberg, Kian Chuan Sia, Siti Humairah Mohd Rodhi, Zhen Ying Fu, A J Rufaihah, Alfredo Franco-Obregón, Mahesh Choolani, Binny Priya Sesurajan, Poh-San Lai, Shir Ying Lee, Pei Lin Koh, Qingfeng Chen, Shu-Uin Gan, Wei Leong Chew, Citra Nz Mattar
Applications: ELISA
Reactive species: Human
"Abstract:Hematopoietic stem cells (HSC) are important targets for gene modification therapies (GMT) as they originate several serious genetic conditions including the β-haemoglobinopathies. Potentially curative ex vivo GMT pose the barriers of accessibility, myeloablation-associated morbidity and prohibitive cost. In vivo GMT using non-integrating single-strand adeno-associated viral vectors (ssAAV) are a promising alternative that address these challenges directly, although the small ssAAV payload limits the capacity to package much larger gene or base editors. We investigated the feasibility of targeting human HSC in vivo with a dual-ssAAV6 strategy, which in future may be useful to deliver split-intein editing tools to overcome this limitation. We engrafted NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ humice with human hCD45 + CD34 + HSC from transfusion-dependent β-thalassemic patients to test in vivo targeting of hCD45 cells with ssAAV6, then administered 5E+12 genomes/kg of ssAAV6-GFP/ssAAV6-mCherry. Humice showed peak single-transgene expression (GFP + or mCh + ) of 1.96-10.17%, and dual-transgene expression (GFP + mCh + ) of 31.77% in circulating hCD45 + cells. Nested hCD45 + from liver, spleen and bone marrow showed single-and dual-transgene expression of 36.13-68.14% and 21.91-59.44% respectively. Secondary transplantation experiments demonstrated long-term persistence of AAV6-transduced hCD45 cells showing single-and dual-transgene expression of 9.19-60.72% and 7.15-9.19% respectively, with significant increase in expression from circulating cells. Minimal pro-inflammatory cytokine expression was observed following ssAAV6 administration in thalassemia humice compared with humice carrying non-thalassemia HSC. Our model demonstrates the efficiency of in vivo ssAAV6-mediated targeting of thalassaemia HSC, potential long-term survivability of transduced cells, and feasibility of a dual-AAV strategy for gene editing, which offers a promising alternative to ex vivo GMT for β-haemoglobinopathies."
"Article snippet:Anti-AAV6 IgG ELISA (Creative Diagnostics, Shirley, NY, USA) was performed following the manufacturer’s instructions."
Publication (1)
COA
Certificate of Analysis
