513. A Simple Method To Ensure Titer Conservation of AAV2 Vector Preparations Delivered with Stainless Steel Needles
Authors: Elias T. Ketchum, Kevin Kwok, Anthony Ramirez, Junshan Hao, Brian Kruegel, Dominick Vacante, Raymond Bartus, Mehdi Gasmi
Journal: Molecular Therapy (2006)

Ceregene is developing AAV2-based gene therapy vectors encoding nerve growth factor (NGF) (CERE-110) and neurturin (NTN) (CERE-120) for the treatment of Alzheimer's and Parkinson's diseases respectively. Targeted delivery of therapeutic vectors to the central nervous system for these applications requires the use of low volumes, low vector titers, and especially low flow rates. Under these conditions, interactions between the delivery hardware materials and the vector particles in suspension can have a profound impact on the characteristics of the vector preparation. For instance, we found that when AAV2 vectors are delivered by way of a stainless steel needle, vector titer measured by QPCR can drop up to 95% from its initial concentration. Reduction in titer is maximal when low concentration preparations (2x1010vg/mL) are delivered at a low flow rate (0.5μL/min). Since stainless steel is the most common material used to manufacture surgical needles, we developed a technique to prevent vector titer reduction without the need for chemicals or protein additives in the vector formulation. This technique consists of pre-treating the stainless steel needles with an identical vector suspension at a higher concentration (≥4x1012vg/ mL) followed by a rinsing step using the vector at the desired concentration. This pretreatment of the delivery hardware results in the saturation of vector binding sites in the lumen of the needle and prevents further vector loss when the desired dose is delivered. Our data show that AAV2 vector binds surgical grade stainless steel with high affinity and does not leach back into suspension in physiological conditions of temperature and salt concentrations. This priming technique was applied in our pre-clinical in vivo experiments and it is currently used in our phase I clinical trials evaluating the safety and tolerability of our CERE-110 and CERE-120 vectors. Ceregene is developing AAV2-based gene therapy vectors encoding nerve growth factor (NGF) (CERE-110) and neurturin (NTN) (CERE-120) for the treatment of Alzheimer's and Parkinson's diseases respectively. Targeted delivery of therapeutic vectors to the central nervous system for these applications requires the use of low volumes, low vector titers, and especially low flow rates. Under these conditions, interactions between the delivery hardware materials and the vector particles in suspension can have a profound impact on the characteristics of the vector preparation. For instance, we found that when AAV2 vectors are delivered by way of a stainless steel needle, vector titer measured by QPCR can drop up to 95% from its initial concentration. Reduction in titer is maximal when low concentration preparations (2x1010vg/mL) are delivered at a low flow rate (0.5μL/min). Since stainless steel is the most common material used to manufacture surgical needles, we developed a technique to prevent vector titer reduction without the need for chemicals or protein additives in the vector formulation. This technique consists of pre-treating the stainless steel needles with an identical vector suspension at a higher concentration (≥4x1012vg/ mL) followed by a rinsing step using the vector at the desired concentration. This pretreatment of the delivery hardware results in the saturation of vector binding sites in the lumen of the needle and prevents further vector loss when the desired dose is delivered. Our data show that AAV2 vector binds surgical grade stainless steel with high affinity and does not leach back into suspension in physiological conditions of temperature and salt concentrations. This priming technique was applied in our pre-clinical in vivo experiments and it is currently used in our phase I clinical trials evaluating the safety and tolerability of our CERE-110 and CERE-120 vectors.