Vector Production Methods Research Articles

Kinematic analysis of intelligent petrochemical fluid handling arm

In view of the low degree of automation of fluid transfer device in China's petrochemical industry, 
 this paper designs and develops an intelligent fluid transfer operator arm instead of manual 
 operation and draws its three-dimensional model based on the common structure type of 
 land-based fluid loading and unloading arm in the current market. The kinematic model of the 
 arm was developed based on the Denavit-Hartenberg method, and the forward and inverse 
 kinematics were analyzed; then the Jacobi matrix of the arm was obtained based on the 
 chi-square transformation matrix and vector product method. Finally, the positive kinematic 
 simulation was carried out by using the Robotics Toolbox toolkit in Matlab software, and the 
 conclusion that the linkage coordinate system established by the D-H method is consistent with 
 the actual structure of the manipulator arm in this paper.

Measurement Method of Human Lower Limb Joint Range of Motion Through Human-Machine Interaction Based on Machine Vision.

To provide stroke patients with good rehabilitation training, the rehabilitation robot should ensure that each joint of the limb of the patient does not exceed its joint range of motion. Based on the machine vision combined with an RGB-Depth (RGB-D) camera, a convenient and quick human-machine interaction method to measure the lower limb joint range of motion of the stroke patient is proposed. By analyzing the principle of the RGB-D camera, the transformation relationship between the camera coordinate system and the pixel coordinate system in the image is established. Through the markers on the human body and chair on the rehabilitation robot, an RGB-D camera is used to obtain their image data with relative position. The threshold segmentation method is used to process the image. Through the analysis of the image data with the least square method and the vector product method, the range of motion of the hip joint, knee joint in the sagittal plane, and hip joint in the coronal plane could be obtained. Finally, to verify the effectiveness of the proposed method for measuring the lower limb joint range of motion of human, the mechanical leg joint range of motion from a lower limb rehabilitation robot, which will be measured by the angular transducers and the RGB-D camera, was used as the control group and experiment group for comparison. The angle difference in the sagittal plane measured by the proposed detection method and angle sensor is relatively conservative, and the maximum measurement error is not more than 2.2 degrees. The angle difference in the coronal plane between the angle at the peak obtained by the designed detection system and the angle sensor is not more than 2.65 degrees. This paper provides an important and valuable reference for the future rehabilitation robot to set each joint range of motion limited in the safe workspace of the patient.

Overview of major AAV production platforms

Jote BulchaUniversity of Massachusetts Medical School“The current AAV-based viral vector production methods cannot meet demand for conducting large-scale clinical trials and commercial applications. Therefore, there is an urgent need to develop innovative AAV production schemes to turn the promise of gene therapy treatments into a reality.”

The Effects of Conditioning and Lentiviral Vector Pseudotype on Short- and Long-Term Airway Reporter Gene Expression in Mice.

A gene addition therapy into the conducting airway epithelium is a potential cure for cystic fibrosis lung disease. Achieving sustained lung gene expression has proven difficult due to the natural barriers of the lung. The development of lentiviral (LV) vectors pseudotyped with viral envelopes that have a natural tropism to the airway has enabled persistent gene expression to be achieved in vivo. The aims of this study were to compare the yields of hemagglutinin (HA) and vesicular stomatitis virus-glycoprotein (VSV-G) pseudotyped HIV-1 vectors produced under the same conditions by our standard LV vector production method. We then sought to measure gene expression in mouse airways and to determine whether lysophosphatidylcholine (LPC) conditioning enhances short- and long-term gene expression. C57Bl/6 mouse airways were conditioned with 10 μL of 0.1% LPC or saline control, followed 1 h later by a 30 μL dose of an HA or VSV-G pseudotyped vector carrying either the LacZ or luciferase reporter genes. LacZ expression was assessed by X-gal staining after 7 days, while lung luminescence was quantified regularly for up to 18 months by bioluminescent imaging. The HA pseudotyped vectors had functional titers 25 to 60 times lower than the VSV-G pseudotyped vectors. Conditioning the lung with LPC significantly increased the total number of LacZ-transduced cells for both pseudotypes compared to saline control. Regardless of LPC conditioning, the VSV-G pseudotype produced higher initial levels of gene expression compared to HA. LPC conditioning did not increase the number of transduced basal cells for either pseudotype compared to saline, and was not required for long-term gene expression. Both pseudotyped vectors effectively transduced the upper conducting airways of wild-type mice. The use of LPC conditioning before vector delivery was not required in mouse lungs to produce long-term gene expression, but did improve short-term gene expression.

TESSA technology: a new paradigm in AAV manufacturing

<!--StartFragment--> <h3>Watch the video or read the poster to learn:</h3> <ul><li>Benefits and drawbacks of current AAV viral vector production methods</li><li>How Tetracycline Enabled Self Silencing Adenovirus (TESSA) allows efficient, scalable AAV manufacture</li><li>Results from studies comparing TESSA with conventional methods</li></ul> <div class="authors authors-lg"><div class="authors authors-lg"><div class="author"><div class="author-img"><img src="https://bioinsights.blob.core.windows.net/uploads/Ryan Cawood 2 (003)-sq.jpg" data-image="1"></div><div class="author-det"><h3 class="author-name">About the speaker</h3> <b>Ryan</b>&nbsp;<b>Cawood</b> founded Oxford Genetics in 2011, after earning a first class degree in genetics and a PhD from Oxford University. The idea behind the company was to simplify and standardise the process of DNA engineering using a proprietary DNA plasmid platform called SnapFast™ that allowed researchers – for the first time – to assemble complex sections of DNA as simply as ‘molecular Lego’. Ryan used his background in genetic engineering and virology to guide and grow the business through a series of strategic changes that explored how further development of the SnapFast™ platform through in house research and development could help overcome multiple challenges in the development of new biologics. This culminated in a rebrand to OXGENE in 2019, as the company redefined itself as a leading solutions provider, using a combination of proprietary technologies to address multiple pinch-points on the journey through design, discovery, development and manufacture of a novel biologic. </div></div></div></div> <!--EndFragment-->

Micro-dystrophin AAV Vectors Made by Transient Transfection and Herpesvirus System Are Equally Potent in Treating mdx Mouse Muscle Disease

Vector production scale-up is a major barrier in systemic adeno-associated virus (AAV) gene therapy. Many scalable manufacturing methods have been developed. However, the potency of the vectors generated by these methods has rarely been compared with vectors made by transient transfection (TT), the most commonly used method in preclinical studies. In this study, we blindly compared therapeutic efficacy of an AAV9 micro-dystrophin vector generated by the TT method and scalable herpes simplex virus (HSV) system in a Duchenne muscular dystrophy mouse model. AAV was injected intravenously at 5 × 1014 (high), 5 × 1013 (medium), or 5 × 1012 (low) viral genomes (vg)/kg. Comparable levels of micro-dystrophin expression were observed at each dose in a dose-dependent manner irrespective of the manufacturing method. Vector biodistribution was similar in mice injected with either the TT or the HSV method AAV. Evaluation of muscle degeneration/regeneration showed equivalent protection by vectors made by either method in a dose-dependent manner. Muscle function was similarly improved in a dose-dependent manner irrespective of the vector production method. No apparent toxicity was observed in any mouse. Collectively, our results suggest that the biological potency of the AAV micro-dystrophin vector made by the scalable HSV method is comparable to that made by the TT method.

High-Titer Production of HIV-Based Lentiviral Vectors in Roller Bottles for Gene and Cell Therapy.

Lentiviral vectors are becoming preferred vectors of choice for clinical gene therapy trials due to their safety, efficacy, and the long-term gene expression they provide. Although the efficacy of lentiviral vectors is mainly predetermined by the therapeutic genes they carry, they must be produced at high titers to exert therapeutic benefit for in vivo applications. Thus, there is need for practical, robust, and scalable viral vector production methods applicable to any laboratory setting. Here, we describe a practical lentiviral production technique in roller bottles yielding high-titer third-generation lentiviral vectors useful for in vivo gene transfer applications. CaPO4-mediated transient transfection protocol involving the use of a transfer vector and three different packaging plasmids is employed to generate lentivectors in roller bottles. Following clearance of cellular debris via low-speed centrifugation and filtration, virus is concentrated by high-speed ultracentrifugation over sucrose cushion.

Small and Micro-Scale Recombinant Adeno-Associated Virus Production and Purification for Ocular Gene Therapy Applications.

Over the past two decades recombinant adeno-associated virus (rAAV) vectors have emerged as the gold standard for transferring genetic material to cells of the retina. The ability to effectively produce small batches of rAAV vector at high enough purity for in vitro and in vivo applications in a cost-effective manner is paramount. This is particularly the case when conducting preclinical experiments to screen novel serotypes, promoters or transgenes, where production of numerous vector batches is required. Current vector production methods often produce large quantities of vector, limiting the cost-effectiveness and practicality of such screening experiments, which often require only small volumes of vector to carry out. Herein, we describe a method to produce high titer (1012-1013 vector genomes (vg)/mL) rAAV vector on small (~100μL) or micro (~15μL) scale for in vitro and in vivo applications.

Research on Kinematics of Assistant Robot Based on Vector Product Method

To the world population aging serious problem, a research on assistant robot is more and more attention. Assistant Robot is a device that is similar to human exoskeleton. This device is worn by the operator, and keep consistent with the human walking and replace body to undertake cargo load. In this paper, based on the method of vector product, a 3-DOF assistant robot’s motion is analyzed. Jacobi matrix of robot motion is calculated. According to the experimental data from human walking, the assistant robot’s displacement and velocity vector and variation are simulated, and its variation are also analyzed. In theory, it proved that the velocity and displacement curves are continuous and smooth. Besides, it also provides references for control system design of the assistant robot.

Viral vector-based models of Parkinson's disease.

In order to study the molecular pathways of Parkinson's disease (PD) and to develop novel therapeutic strategies, scientific investigators rely on animal models. The identification of PD-associated genes has led to the development of genetic PD models as an alternative to toxin-based models. Viral vector-mediated loco-regional gene delivery provides an attractive way to express transgenes in the central nervous system. Several vector systems based on various viruses have been developed. In this chapter, we give an overview of the different viral vector systems used for targeting the CNS. Further, we describe the different viral vector-based PD models currently available based on overexpression strategies for autosomal dominant genes such as α-synuclein and LRRK2, and knockout or knockdown strategies for autosomal recessive genes, such as parkin, DJ-1, and PINK1. Models based on overexpression of α-synuclein are the most prevalent and extensively studied, and therefore the main focus of this chapter. Many efforts have been made to increase the expression levels of α-synuclein in the dopaminergic neurons. The best α-synuclein models currently available have been developed from a combined approach using newer AAV serotypes and optimized vector constructs, production, and purification methods. These third-generation α-synuclein models show improved face and predictive validity, and therefore offer the possibility to reliably test novel therapeutics.

On the arithmetic operations over finite fields of characteristic three with low complexity

In this paper, the Hermite polynomial representation is adapted as a new way to represent certain finite fields of characteristic three. We give the multiplication method to multiply two elements of F3n in the Hermite polynomial representation with subquadratic computational complexity by using a divide-and-conquer idea. We show that in some cases there is a set of irreducible binomials in the Hermite polynomial representation to obtain modular reduction with a lower addition complexity than the standard polynomial representation. We also investigate the matrix vector product method for the multiplication of the field elements represented by Hermite polynomials.

An Analysis and Investigation of Algorithms for Identifying Convexity-Concavity of a Simple Polygon

Algorithms for identifying convexity-concavity of a simple polygon has a very important application in many fields.The authors analyzed the present popular algorithms for identifying convexity-concavity of a simple polygon such as angling method,left-right-point method,vector-area method,vector-product method,raying method,slopping method and extremity-vertices-order method.A detailed derivation of these algorithms has revealed that these algorithms can all use the formula b=p*m as the expression,and are equivalent to each other in nature;nevertheless,the pole-order method still have some problems to be further studied.Based on an analysis of the computation,the authors hold that theoretically the vector-product method,the slopping method and the raying method could be used effectively in programming.

Development of a recombinant adenovirus vector production system free of replication-competent adenovirus by utilizing a packaging size limit of the viral genome

In a conventional adenovirus (Ad) vector production method using 293 cells, homologous recombination between Ad vector DNA and 293 cell-derived Ad E1 DNA occurs with low efficiency, resulting in the generation of replication-competent adenovirus (RCA). RCA can induce the spread of replication-incompetent Ad vectors, leading to unexpected tissue damage. In order to overcome this problem, we developed an Ad vector production system free of RCA generation by utilizing the Ad packaging size limit of the viral genome. It is well known that up to approximately 105% (37.7 kb) of the wild-type genome (35.9 kb) can be packaged in the Ad virion. We designed the Ad vector genome by insertion of a transgene expression cassette into the E3 region, such that homologous recombination between the Ad vector DNA and 293 cell-derived Ad E1 DNA would produce an Ad vector genome that exceeds in the size of the packaging limit. In accord with our strategy, no RCA generation was observed during the passages when we used the E1 (3.2 kb)-deleted Ad vectors containing a more than 3.0-kb transgene expression cassette in the E3 region. In contrast, the E1 (3.2 kb)-deleted Ad vectors, which retain 37.7 kb of the viral genome and have an insertion of a 2.1-kb transgene expression cassette in the E3 region, generated RCA, although RCA derived from this Ad vector exceeded the packaging size limit (105.0%). These results suggest that RCA generation can be avoided when the genome size of RCA is more than 108.3% (38.9 kb) of the wild-type Ad genome. This Ad vector production system generates safe, easy, and efficient Ad vector stock for both basic study as well as clinical research.

A versatile adeno-associated virus vector producer cell line method for scalable vector production of different serotypes.

Application of adeno-associated virus (AAV) vector in large animal studies and clinical trials often requires high-titer and high-potency vectors. A number of currently used vector production methods, based on either transient transfection or helper virus infection of cell lines, have their advantages and limitations. We previously developed a 293-cell-based producer cell line method for high-titer and high-potency AAV2 vectors. Similar to several other methods, however, it requires multiple cloning steps for the vector and packaging plasmids and a two-step transfection and selection for stable cell lines. Here we report a simplified method with several key improvements and advantages: (1) a one-step cloning of AAV vector cassette into the serotype-specific packaging plasmid; (2) a single plasmid transfection and selection for stable AAV vector producer cell lines; (3) high vector yields of different serotypes, e.g., AAV2, 8, and 9, upon infection with an E1A/E1B-deleted helper adenovirus; (4) efficient packaging of both single-stranded and double-stranded (self-complementary) AAV vectors; and (5) efficient packaging of large AAV cassettes such as a mini-dystrophin vector (5.0 kb). All cell lines were stable with growth rates identical to the parental 293 cells. The vector yields were consistent among serotypes, with 5 × 10(13) to 8 × 10(13) vector genome particles per Nunc cell factory (equivalent to 40 15-cm plates). The vectors showed high potency for in vitro and in vivo transduction. In conclusion, the simple and versatile AAV producer cell line method can be useful for large scale AAV vector production in preclinical and clinical studies.

Antitumor Effects of Targeting hTERT Lentivirus-Mediated RNA Interference Against KB Cell Lines

Squamous cell carcinomas are the leading frequent malignant tumors in the oral and maxillofacial region. Currently available treatment options are of limited efficacy, and there is an urgent need for development of alternative therapies. RNA interference (RNAi) is a sequence-specific RNA degradation process. In this study, we screened and identified an in vitro-transcribed 21-bp shRNA targeting human telomerase reverse transcriptase (hTERT) from three candidates and generated a lentivirus vector. Subsequent experiments indicated that this lentiviral transgenic system could effectively transfer into target KB cells, above 80% gene transfer efficiency at MOI of 2.5, and significantly and specifically inhibited hTERT expression both in mRNA (73.42%) and protein (74.67-82.91%) levels. To further evaluate the role of hTERT-targeted RNAi, we found that hTERT inhibition consequently induced suppression of cyclin D1 (54.67%), upregulation of caspase-3 (100.10%), and caspase-9 (42.67%) of KB cells. Therefore, the apoptosis rates of KB cells were increased by 206.33%. In conclusion, these data indicated the potential of lentivirus vectors in cancer gene therapy, especially after development of more efficient vector production methods, and higher virus titers demonstrated that targeting hTERT RNAi may result in telomere uncapping, which triggers cell cycle arrest and apoptosis signal and leads to tumor suppression.

A GUTLESS ADENOVIRAL VECTOR EXPRESSING FULL‐LENGTH ANTI‐Her2 ANTIBODY

1. Therapeutic monoclonal antibodies are increasingly being used in clinical cancer treatment, but their complex technology and high cost limit their use. Helper-dependent (HD) adenoviruses are among the most efficient and safe gene therapy vectors capable of mediating long-term expression. 2. Using Gateway (Invitrogen, San Diego, CA, USA) cloning technology, we constructed an HD–trastuzumab (TAb) plasmid carrying the full-length anti-HER2 antibody gene. Using an efficient recombinase, namely in vitro-evolved Flippase-expressing recombinase, to excise the helper virus packaging signal in producer cells, we developed a scalable HD vector production method. Antibody expression of HD-TAb in vitro was detected by ELISA and western blot. 3. The full-length antibody gene delivery system allowed for continuous production of a full-length antibody at a high concentration. Bioactive antibody macromolecules were generated via gene transfer in vitro. 4. In conclusion, HD adenoviral vectors can stably express a full-length antibody for prolonged periods without the difficulties associated with sophisticated antibody manufacture techniques and at a much lower cost. As a promising tool for gene therapy, this novel system can shorten the duration and reduce the expense of antibody development.

Immunosuppression Modulates Immune Responses to AAV Capsid in Human Subjects Undergoing Intramuscular Gene Transfer for Lipoprotein Lipase Deficiency

Administration of adeno-associated viral vectors (AAV) has resulted in long-term therapeutic gene transfer in multiple large animal models of disease, but attempts to translate systemic administration of AAV to humans have been limited in some cases by an immune response to the vector capsid (Nature Med 12:342–7, 2006; Nature Med 13:419–422, 2007). To overcome this obstacle, we have proposed that a short course of immunosuppression (IS) be administered with vector injection. Here we report the safety and efficacy results of this maneuver in a trial of AAV-1 administered to skeletal muscle. Lipoprotein lipase (LPL) deficiency is a familial disorder in which insufficient levels of LPL enzyme result in the accumulation of triglycerides in plasma. In a clinical study to correct this disorder, an AAV-1 vector encoding the therapeutic transgene LPL was administered to the skeletal muscle of affected individuals. Eight subjects were assigned to two dose cohorts, receiving 1×1011 genome copies (gc)/kg or 3×1011gc/kg. In this study, one subject receiving the high vector dose experienced a transient, asymptomatic increase in the muscle enzyme creatinine phosphokinase beginning 4 weeks after gene transfer, persisting for several weeks. This was associated with capsid-specific CD4+ and CD8+ T cell activation detectable by IFN-γ ELISPOT and intracellular cytokine staining on PBMC. In total, a T cell response to the AAV capsid, but not to the LPL transgene, was detectable in 4/8 subjects. In some of these subjects, T cell responses were detectable in peripheral blood up to 2 years after gene transfer. To prevent potentially harmful immune responses directed to the AAV capsid, a follow up study in LPL deficient subjects was initiated in which a 12-week regimen of mycophenolate mofetil and cyclosporine A was administered orally starting at the time of AAV-1 intramuscular gene transfer. Two additional subjects were administered AAV-1-LPL in the absence of immunosuppression, to compare the safety and efficacy of two different vector production methods. Overall, IS was well tolerated and no adverse events were reported. At a dose of 3×1011 gc/kg, IS effectively blocked T cell responses to capsid, which were undetectable by IFN-γ ELISPOT in 4/4 subjects, even after IS was discontinued. However, at a dose of 1×1012gc/kg, a delayed IFN-γ response to capsid antigen was observed in 3/5 subjects. In two subjects the T cell response was still detectable after IS was discontinued. T cell responses did not correlate with pre-existing antibody titers in any of the subjects, as positivity for antibodies against the AAV capsid was not predictive of ELISPOT results. Antibody analysis revealed that IS did not have any effect on the development of antibodies against AAV-1 capsid, as all subjects developed humoral immunity against capsid, with predominance of IgG1 antibody subclass. None of the subjects receiving IS developed humoral or cellular immunity to the LPL transgene product. In conclusion, the use of IS in the context of AAV-1 gene transfer for LPL deficiency is safe and at least partially effective in blocking T cell responses directed to the capsid antigen. Ongoing long-term evaluation of transgene expression in these subjects will allow further assessment of the effects of IS on efficacy of gene transfer.

A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy

BackgroundDuchenne muscular dystrophy (DMD) is an X-linked recessive disorder with monogenic mutations setting the stage for successful gene therapy treatment. We have completed a study that directly deals with the following key issues that can be directly adapted to a gene therapy clinical trial using rAAV considering the following criteria: 1) A regional vascular delivery approach that will protect the patient from widespread dissemination of virus; 2) an approach to potentially facilitate safe passage of the virus for efficient skeletal muscle transduction; 3) the use of viral doses to accommodate current limitations imposed by vector production methods; 4) and at the same time, achieve a clinically meaningful outcome by transducing multiple muscles in the lower limb to prolong ambulation.MethodsThe capacity of AAV1, AAV6 or AAV8 to cross the vascular endothelial barrier carrying a micro-dystrophin cDNA was compared under identical conditions with delivery through a catheter placed in the femoral artery of the mdx mouse. Transduction efficiency was assessed by immuno-staining using an antibody (Manex1a) that recognizes the N-terminus of micro-dystrophin. The degree of physiologic correction was assessed by measuring tetanic force and protection from eccentric contraction in the extensor digitorum longus muscle (EDL). The vascular delivery paradigm found successful in the mouse was carried to the non-human primate to test its potential translation to boys with DMD.ResultsRegional vascular delivery resulted in transduction by rAAV8.micro-dystrophin reaching 94.5 ± 0.9 (1 month), 91.3 ± 3.1 (2 months), and 89.6 ± 1.6% (3 months). rAAV6.micro-dystrophin treated animals demonstrated 87.7 ± 6.8 (1 month), 78.9 ± 7.4 (2 months), and 81.2 ± 6.2% (3 months) transduction. In striking contrast, rAAV1 demonstrated very low transduction efficiency [0.9 ± 0.3 (1 month), 2.1 ± 0.8 (2 months), and 2.1 ± 0.7% (3 months)] by vascular delivery. Micro-dystrophin delivered by rAAV8 and rAAV6 through the femoral artery significantly improved tetanic force and protected against eccentric contraction. Mouse studies translated to the hindlimb of cynamologous macaques using a similar vascular delivery paradigm. rAAV8 carrying eGFP in doses proportional to the mouse (5 × 1012 vg/kg in mouse vs 2 × 1012 vg/kg in monkey) demonstrated widespread gene expression [medial gastrocnemius – 63.8 ± 4.9%, lateral gastrocnemius – 66.0 ± 4.5%, EDL – 80.2 ± 3.1%, soleus – 86.4 ± 1.9%, TA – 72.2 ± 4.0%.ConclusionThese studies demonstrate regional vascular gene delivery with AAV serotype(s) in mouse and non-human primate at doses, pressures and volumes applicable for clinical trials in children with DMD.

Adeno-associated Virus Type 5 Reduces Learning Deficits and Restores Glutamate Receptor Subunit Levels in MPS VII Mice CNS

A major challenge in treating lysosomal storage diseases with enzyme therapy is correcting symptoms in the central nervous system (CNS). This study used a murine model of mucopolysaccharidosis type VII (MPS VII) to test whether pathological and functional CNS defects could be corrected by expressing beta-glucuronidase via bilateral intrastriatal injection of adeno-associated virus type 5 (AAV5betagluc) vectors. After injecting AAV5betagluc, different brain regions expressed active beta-glucuronidase, which corrected lysosomal storage defects. Compared to age-matched littermates, adult MPS VII mice were impaired in spatial learning and memory, as measured by the repeated acquisition and performance chamber (RAPC) assay. AAV5betagluc-treated MPS VII mice improved significantly in the RAPC assay, relative to saline-injected littermates. Moreover, our studies reveal that cognitive changes in MPS VII mice correlate with decreased N-methyl-d-aspartate and alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid receptor expression. Importantly, AAV5betagluc delivery restored glutamate receptor levels. Together, these data demonstrate that AAV5 vectors deliver a therapeutically effective beta-glucuronidase gene to the CNS and further suggest a possible mechanism underlying spatial learning defects in MPS VII mice.

503. A Novel Herpes Simplex Virus Helper Based Production of Adeno- Associated Virus Vectors for Treatment of Retinal Angiogenesis

AGTC's proprietary rAAV vector production method uses recombinant herpes simplex virus (HSV) as the helper agent for rAAV production. This production method has been demonstrated to be both highly scalable and productive compared to other rAAV production methods such as the transient co-transfection or adenovirus co-infection process. rAAV production by rHSV-1 methodology requires two recombinant HSV-1 vectors, rHSV-1- rep2cap2 which expresses the AAV 2 rep and cap genes, and a second rHSV-1 that contains the therapeutic gene of interest flanked by the AAV-2 inverted terminal repeats.Both rHSV vectors are deleted in an essential gene for HSV replication and hence they are propagated only in a complementing cell line. Here we report the construction and characterization of rHSV-TR-shFlt-1 that contains a novel soluble hybrid version of Flt-1, the receptor for vascular endothelial growth factor (VEGF). This novel shFlt-1 is a high-affinity binder of VEGF, a central player in pathologic neovascularization in both wet age-related macular degeneration (AMD) and proliferative diabetic retinopathy (PDR). To construct rHSV-TR-shFlt-1, a shuttle plasmid containing the expression cassette of sFlt-1 flanked with homologous HSV-1 sequences was first constructed and co-transfected with rHSVd27.1- TR-GFP viral DNA into complementing V27 cells. rHSV-TR-shFlt-1 was screened against the parental rHSV-1-GFP viruses that express fluorescent GFP, and the homologous recombination was further confirmed by Southern blot analysis. Selected rHSV-TR-shFlt-1 clones were propagated through 10 passages and their ability to produce rAAV2-TR-shFlt-1 vector was examined by real-time PCR. The expression level, size, and in-vitro biological activity of sFlt-1 expressed in rAAV2-TR-shFlt-1 transduced cells were determined by ELISA, immunoblot, and the HUVECs proliferation assay respectively. Stability studies indicated that the rHSV-TR-shFlt-1 helpers maintained their ability to produce rAAV comparable to that of the positive control rHSV-1-GFP through 10 passages. Preliminary data from the oxygen-induced retinopathy (OIR) mouse model suggests that rAAV2-shFlt-1 derived from rHSV1- TR-shFlt-1 is able to inhibit retinal neovascularization, and there is no significant difference compared to rAAV2-shFlt-1 vector made by the transfection method. To our knowledge this is the first report of the construction and characterization of a recombinant HSV helper virus to produce a rAAV vector encoding a therapeutic gene for inhibition of ocular angiogenesis. Further preclinical evaluation of the AAV2-shFlt-1 is in the process.