670 increased therapeutic effect of cell transplantation for wound healing in diabetic mice by prolonging the survival of transplanted cells using adhesamine, a synthetic adhesion molecule

670 Increased Therapeutic Effect of Cell Transplantation for Wound Healing in Diabetic Mice by Prolonging the Survival of Transplanted Cells Using Adhesamine, a Synthetic Adhesion Molecule Molecular T[.]

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OLIGONUCLEOTIDE & RNAI THERAPEUTICS II

666 Nanoparticles are Effective Vehicles

for Systemic Delivery of 2’OMePS Antisense

Oligonucleotides in Exon Skipping-Mediated

Dystrophin Restoration

Alessandra Ferlini,1 Marina Fabris,1 Elena Bassi,1 So a Falzarano,1

Daniela Perrone,2 Francesca Gualandi,1 Patrizia Sabatelli,1,3

Luciano Merlini,1 Paola Rimessi.1

1 Department of Experimental and Diagnostic Medicine, Section of

Medical Genetics, University of Ferrara, Ferrara, Italy; 2

IGM-C.N.R., Unit of Bologna, IOR, Bologna, Italy; 3 Department of

Chemistry, University of Ferrara, Ferrara, Italy.

We used a novel PMMA/N-isopropil-acrylamide+ (NIPAM)

cationic nanoparticle (ZM2-NPs), found to be non-toxic in vitro,

for systemic delivery of 2’-O-methyl full-length phosphorothioate

AONs in the mdx animal model Six weeks-old mdx mice were

intraperitoneally treated: group 1 received 225 mg of naked M23D

AON and group 2 received of 225 mg of M23D AON conjugated

with 2.5 mg of ZM2 NPs Four non-injected mdx mice were used

as controls The animals were injected weekly for 7 weeks (7.5 mg/

kg/injection) and sacri ced 1 week (4) and 12 weeks (4) after the

 nal administration The total amount of M23D AON received by

each animal was 52.5 mg/kg (1575 mg/animal) In treated animals

sacri ced 1 week after  nal administration we observed restored

dystrophin protein synthesis in both skeletal (quadriceps, diaphragm)

and cardiac muscles, allowing protein localization in up to 40%

of muscle  bers The mdx exon 23 skipping level was up to 20%

Western blotting showed the dystrophin protein in all muscles

studied Furthermore, we veri ed that dystrophin restoration also

occurs in the smooth muscle cells of the dorsal skin arrector pili in

ZM2-AON treated mdx mice only In treated animals sacri ced 12

weeks after  nal administration we still observed dystrophin protein

synthesis in up to 8% of muscle  bers in skeletal muscle (quadriceps,

diaphragm) but not in the heart The mdx exon 23 skipping level was

6% (quadriceps), 8% (heart) and 10% (diaphragm) Western blotting

revealed the presence of dystrophin protein in the diaphragm only

We measured the dystrophin transcript amount in all animal groups

by four exon speci c real time PCR assays (ESRAs) In non-treated

mice, we found that the dystrophin transcript amount ranged from

20 to 30% in all muscles of younger mice (7 weeks of age), whereas

it was lower in skeletal muscles (10%-20%) and higher in the heart

(up to 30%) in older mice (19 weeks of age) In ZM2-AON or naked

AON treated animals sacri ced 1 week after  nal administration, the

dystrophin transcript amount didn’t vary signi cantly Differently,

in 12 weeks/sacri ced ZM2-AON treated animals, the transcript

level was signi cantly higher in skeletal muscles but reduced in the

heart In mice treated with naked AONs the amount of transcript was

higher also in the heart In conclusions, ZM2-AON complexes are

able to induce dystrophin restoration, which still persists in skeletal

muscles, though at low level, 12 weeks after  nal administration

Dystrophin transcript amount is not in uenced by naked AON or

NP-AON therapy in early sacri ced mice, whereas both naked AONs

and ZM2-AONs seem to enhance the dystrophin transcript stability

in late sacri ced mice when the basal transcription level is lower The

basal dystrophin transcription amount in the cardiac muscle seems to

be age-related, having a different behavior during AON therapy

667 Therapy for Vaso-Occlusion in Sickle Cell Disease Includes RNA Aptamer

Angela D Burnette,1 Jun-ichi Nishimura,2 Milena Batchvarova,3

Shahid M Nimjee,1 Rahima Zenadi,3 Marilyn J Telen,3 Bruce A

Sullenger.1

1 Surgery, Duke University Medical Center, Durham, NC;

2 Hematology/Oncology, Osaka University Graduate School of Medicine, Osaka, Japan; 3 Hematology/Oncology, Duke University

Medical Center, Durham, NC.

Painful vaso-occlusive episodes are characteristic of sickle cell disease (SCD) and are caused in part by the adhesion of sickle erythrocytes (SS-RBC) to the vascular endothelium As a possible treatment for vaso-occlusion to inhibit SS-RBC adhesion, two adhesion molecules: αvβ3 and P-selectin, were targeted by

high-af nity aptamers and selected via SELEX (Systematic Evolution of Ligands through EXponential enrichment) technology An in vitro

 ow chamber was used to test the anti-adhesion activity of aptamer 17.16 that binds to human integrin αvβ3 Using human umbilical vein endothelial cells (HUVEC) pre-treated with thrombin, human SS-RBC were passed across the HUVEC at a constant rate Aptamer 17.16 at 30nM had anti-adhesion activity similar to LM609, an inhibitory antibody to αvβ3 However, a control aptamer did not inhibit adhesion Normalized % inhibition of 30nM aptamer 17.16

at 2 dynes/cm2 shear stress was 68%

The anti-adhesive activity of aptamer PF377, which binds to human P-selectin, was tested using primary HUVEC pre-treated with IL-13, followed by histamine Aptamer PF377 at 60nM had anti-adhesion activity similar to heparin, a known inhibitor of SS-RBC adhesion to P-selectin, and 9E1, an inhibitory antibody to P-selectin Normalized

% inhibition of 60nM aptamer PF377 at 1 dyne/cm2 shear stress was 99% Negative controls, a functional aptamer and AC1.2, a

non-inhibitory antibody to P-selectin, did not prevent adhesion

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CELL PROCESSING AND VECTOR PRODUCTION

These data show the potential of using aptamers to block adhesion molecules as a viable therapeutic option for preventing vaso-occlusion

in SCD

Cell Processing and Vector Production

668 GMP Production of Self-Complementary Serotype 8 AAV Vector for Treatment of Hemophilia B

James A Allay,1 John S Coleman,1 Arthur W Nienhuis,2 Andrew Davidoff,3 Amit C Nathwani,4 Susan Sleep,1 John T Gray.2

1 Children’s GMP, LLC, Memphis, TN; 2 Hematology, St Jude Children’s Research Hospital, Memphis, TN; 3 Surgery, St Jude Children’s Research Hospital, Memphis, TN; 4 UCL Cancer Institute, University College, London, United Kingdom.

We utilized a 293T-based 2-plasmid transient transfection system coupled with a 3 column chromatography puri cation process to produce a high quality self-complementary AAV2/8 hFIX clinical-grade vector for treatment of Hemophilia B Yields of >2x1012 /10-stack cell factory were obtained and produced ∼3.8x1014 total vector genomes (vg) by QPCR Capsid westerns of all transfected cell lysates allowed continuous monitoring of the transfection productivity Benzonase-treated mirco uidized lysates generated from pellets of transfected cells were puri ed by group separation

on sepharose beads followed by anion exchange chromatography

The virus-containing fractions were further processed through gel

 ltration and ultra ltration using a 100kd membrane The vector was formulated in phosphate-buffered saline plus 0.25% human serum albumin The preparation was free of any adventitious agents

Spectrophotometric analysis suggests ∼20% full particles while only very low quantities of non-viral proteins are visible on silver-stained SDS PAGE Residual 293T DNA and protein were 1.15 pg/109 vg and 0.024 µg/ml, respectively Residual plasmid DNA was 1.1% of the total DNA and residual capsid DNA was 0.013% An infectious assay to measure replicative forms of AAV (rcAAV) that may be generated during production was developed in 293T cells using an E1, E3 deleted adenovirus co-infection to provide the requisite helper function QPCR of capsid sequences after 3 successive rounds of ampli cation indicated the quantity of the rcAAV contaminant is less than or equal to 103 vg per 2.25x1010 vg of vector, which equates to

≤ 1 rcAAV in 2.25x107 vg Additional studies have con rmed the long term stability of the vector at -80°C for at least 30 months and for at least 24 hours formulated in the clinical diluent and stored at room temperature within i.v bags This material has been approved

for use in clinical trials in the US and UK The approved clinical trial protocols are currently open and actively recruiting patients To our knowledge this represents the  rst clinically approved AAV vector preparation using either serotype 8 capsid or a self-complementary genome con guration

669 Production of Lentiviral Vectors

in Suspension Cells Using Recombinant Baculoviruses

Hanna P Lesch, Anna E Laitinen, Lauri M Laitinen, Jere T Pikkarainen, Haritha Samaranayake, Seppo Ylä-Herttuala, Kari J Airenne

Department of Biotechnology and Molecular Medicine, A.I Virtanen Institute, University of Kuopio, Kuopio, Finland; Department of Medicine and Gene Therapy Unit, University of Kuopio, Kuopio, Finland; Kuopio University Hospital, Kuopio, Finland; Ark Therapeutics, Kuopio, Finland.

The known fact is that the production of lentiviral vectors in clinical scale is demanding So far the dominating production method is a transient plasmid transfection system using adherent 293T cells Earlier, we have developed the method for baculovirus-mediated for lentiviral vector production in adherent cells However, the expansion

of the adherent cell production to large volumes is troublesome The most practical method for large scale production would be to use suspension cells which necessitates the use of bioreactors in viral vector production In this study we have adapted the lentivirus production to suspension cells using four recombinant baculoviruses expressing all elements required for a safe lentivirus vector production

Up to 109 TU/ml lentivirus titers were achieved when baculoviruses were used in the transduction of suspension 293T cells and lentiviruses were concentrated by ultracentrifugation The produced lentiviruses were accurately characterized and compared to the viruses produced either in adherent 293T cells by calcium phosphate transfection or

in suspension 293T cells by polyethylenimine transfection The production of baculoviruses is easy and rapid, their use is safe and they have low levels of cytotoxicity; therefore they offer a new alternative way for the production of lentiviral vectors In conclusion, the baculovirus system is ef cient, safe and enables high titer lentivirus production in suspension cells

670 Increased Therapeutic Effect of Cell Transplantation for Wound Healing in Diabetic Mice by Prolonging the Survival of Transplanted Cells Using Adhesamine, a Synthetic Adhesion Molecule

Tetsuya Suehara,1 Makiya Nishikawa,1 Yuki Takahashi,1 Sayumi Yamazoe,2 Motonari Uesugi,2 Yoshinobu Takakura.1

1 Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan; 2 Institute for Chemical Research, Kyoto University, Kyoto, Japan.

Technology for cell culture and differentiation has greatly increased the possibility of cell-based therapy for a variety of diseases Spatiotemporal distribution of administered cells is an important factor determining the therapeutic effect of such treatment However, the survival of administered cells has not been evaluated well enough

to conclude whether it affects the therapeutic effects of cell-based therapy This is at least partly due to the lack of technologies to increase the availability and survival of cells Recently, Yamazoe

et al have discovered a dumbbell-shaped synthetic small molecule, adhesamine, which increases cell adhesion to culture dishes In the present study, we examined whether adhesamine prolongs the survival of cells after transplantation and increases the therapeutic effects of cell transplantation in mouse models of wound healing To

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CELL PROCESSING AND VECTOR PRODUCTION

quantitatively evaluate the distribution and survival of transplanted

cells, NIH/3T3 cells, a mouse  broblast cell line, were genetically

labeled with  re y luciferase to obtain 3T3/Luc cells Two 8-mm

full-thickness excisional skin wounds were made on the dorsum of

ICR mice after hair removal Then, 3T3/Luc cells pretreated with

or without adhesamine were intradermally transplanted around the

wounds, and the survival of cells and the size of the wounds were

periodically measured 3T3/Luc cells rapidly disappeared from the

administration site, but thosetransplanted with adhesamine were

detected at the site for a much longer period of time Such changes

were not obtained with cells transplanted with collagen or  bronectin

In accordance with the cell survival, 3T3/Luc cells with adhesamine

healed the wound signi cantly faster than untreated cells Similar

results were obtained using primary bone marrow derived cells not

only in ICR mice but in diabetic db/db mice, in the latter of which

wound healing is impaired compared with normal mice These

results strongly suggest that adhesamine can be used to increase

the therapeutic effects of transplanted cells for wound healing by

increasing their survival time after transplantation

671 Characterization of Positioning Effect

of pol III-shRNA Transcription Unit in scAAV

Vector Genome on the Packaging Ef ciency and

Functionality of shRNA Silencing

Jun Xie,1 Ran He,1 Qin Su,1 Guangping Gao.1

1 Gene Therapy Center and Viral Vector Core, UMass Medical

School, Worcester, MA.

Recombinant adeno-associated virus (rAAV) can ef ciently deliver

shRNAs to different animal models and accomplish stable knockdown

of target genes Small sizes of Pol III-shRNA expression cassettes

make them more suitable for self-complementary rAAV (scrAAV)

design which is more ef cient than single stranded vector However,

one of the challenges in production of rAAV-shRNA, particularly,

scrAAV-shRNA, is the significant decrease in vector yields as

compared to the vectors without shRNA inserts We compared vector

yields of scrAAV preparations of different serotypes expressing

different shRNAs with those of scrAAVs without a shRNA cassette

We found that the average yield for 17 lots of scrAAV-shRNA vectors

was only 25% of that for 55 lots of regular scrAAVs while all vector

lots were produced in 1x10e9 of 293 cells by the conventional triple

transfection and CsCl puri cation method In addition, EM analysis

suggested that the vectors carrying shRNAs had signi cant higher

rations of empty to full virions Further analysis indicated that, in

most of those scrAAV-shRNA vectors, shRNA expression cassettes

were cloned into downstream of a Pol II promoter-driven EGFP

reporter gene cassette and at a position immediately adjacent to the 3’

inverted terminal repeat (ITR) We hypothesized that negative impact

of shRNA inserts on the vector yield was due to the close vicinity

between the two strong hairpin structures (i.e replication competent 3’

ITR of the scrAAV and shRNA insert), which hinders vector genome

rescue, replication and/or packaging To test this hypothesis and

characterize possible positioning effect of shRNA in scrAAVs, we

constructed a panel of six scrAAV genomes in which a U6 -shRNA

cassette targeting  re  y luciferase (FFLuc) gene was cloned either

immediately after 5’ crippled ITR or 3’ intact ITR or in an intron

between a Pol II promoter and EGFP cDNA in both directions

respectively To examine functionalities of the U6-FFLucShRNA

expressed from different locations of the vector genome and the

shRNA bearing intron, we transfected these 6 vector and appropriate

control plasmids together with a luciferase expression plasmid into

293 cells to compare their ef ciencies of FFLuc gene silencing and

EGFP expression The results demonstrated that the shRNA cassette

worked equally well at any of 3 positions and both directions Insertion

of shRNA cassette into the intron did not affect EGFP expression

We then compared vector yields and packaging ef ciencies of 6

vectors in side-by-side production runs, leading to the following observations First, among the 3 different shRNA positions, vector yields ranked as Intron ≥ 5’ crippled ITR > 3’ intact ITR Secondly,

we noticed additional 80% drop in yield when the hairpin structure

of shRNA cassette was closer to 3’ ITR which is involved in vector replication Taken together, our results con rmed the positioning effect of shRNA in scrAAV vectors and support our hypothesis that two adjacent hairpin structures affect vector genome replication and packaging ef ciency The  ndings from this study provided some important considerations in scrAAV-shRNA vector design

672 Rapid Production of High Titer Virus Using

a Concentrated Retrovirus Expansion Device

Ceidy Sanchez,1 Gianpietro Dotti,1 John Wilson,2 Cliona M

Rooney,1 Malcolm K Brenner,1 Juan F Vera.1

1 Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children’s Hospital, The Methodist Hospital, Houston, TX;

2 Wilson Wolf Manufacturing Corporation, New Brighton, MN.

One impediment to the successful development of human therapies using genetically modi ed hemopoietic cells is the cost and complexity of vector manufacture, which limits the number of studies that can be performed Retroviral transduction is commonly used for such ex vivo modi cation and usually requires large volumes

of retroviral supernatants, the titers of which are low compared with other viral vectors We now describe the use of a Concentrated

Retrovirus Expansion Device (CRED) for vector production The

CRED is similar in external appearance and shape to conventional

roller bottles, and thus can be accommodated by standard roller racks However an internal dialysis membrane allows virus particles

to become concentrated in the cell compartment, while the nutrient compartment is separated but easily accessible and can retain up

to 250 ml of media without causing virus dilution The CRED has

a surface area of 600cm2 in the cell compartment, which, due to diffusion across the dialysis membrane, requires only 10ml of media for cell survival, a 10-fold lower requirement than a traditional cell factory or roller bottle of equivalent area To test the potency and

stability of CRED-produced retrovirus we cultured a clinical grade

PG-13 producer cell line that produces SFG-CAR-CD19/CD28/Z, which encodes a chimeric antigen receptor directed to CD19 (CD19-CAR) This vector is used in a current clinical protocol to transduce

T cells for use in patients with advanced CD19+ B cell malignancies

We compared our standard procedure for retrovirus production (using

a T175  ask) and 30ml of IMDM against the CRED using 10ml of

IMDM Retroviral supernatant was harvested and frozen daily once cell con uence was attained (days 4-5 for the CRED and days 2-3 for the T175) Subsequently, to enumerate viable viral particles, 1x104 HeLa cells were seeded in a 6 well plate and after 24 hrs were retrovirally transduced using volumes ranging from 10-300ml in the presence of 1ul of Polybrene After 6 days cultures were evaluated by

 ow to assess transduction ef ciency, using an antibody recognizing the CH2CH3 sequence of CAR-CD19/CD28/Z The transduction

ef ciency of the CRED virus was at least 4 times greater than

conventional supernatant; 10ul (7.5%±4.5% vs 2.5%±0.28%), 20ul (11.9%±6.3% vs 4.9%±0.1%), 50ul (25.9%±7.4% vs 7.9%±0.9%), 100ul (45.2%±11.5% vs 9.3%±0.6%), 200ul (75.15%±15.6% vs 16.9%±1.5%), 300ul (85.7%±8.6% vs 21.4±1.8%) (n=4 expts)

To ensure vector stability we exposed the harvested supernatant to

6 consecutive freeze-thaw cycles, and compared virus titers We initially obtained an 86.8% transduction ef ciency of HeLa cells which slowly declined after each cycle (82.4%, then 80.2%, 80%,

68.7%, 60.9% and 55.5%) Hence the CRED can produce large

quantities of high titer retrovirus without media change This virus can resist freeze-thaw cycles and occupies 5 fold less storage space

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CELL PROCESSING AND VECTOR PRODUCTION

than conventional reagents This combination of features should enable this scalable production technique to facilitate clinical studies

of retroviral vectors

673 A Novel Approach to Developing Recombinant AAV Producing Cell Lines

Jingmin Zhou, Katherine A High, Guang Qu

Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia and The University of Pennsylvania, Philadelphia, PA.

Adeno-associated viral vectors have shown promise in ef cient gene delivery to treat human diseases Efforts were made to develop scalable methods to produce recombinant adeno-associated virus (rAAV) as the  eld of rAAV gene therapy continues to move rapidly

to clinical application In addition to the widely used HEK 293 triple-transfection method, more easily scalable methods, cell lines and baculovirus-based AAV production systems have been developed

in the past several years All of these production methods have been used to manufacture pre-clinical and clinical AAV vectors In this study, we present the preliminary data of a novel approach to develop rAAV producing cell lines We hypothesized that high copy number of rAAV genomes integrated into the cell lines may enhance rAAV yield and that high ratio of rAAV vector genomes to the rep/

cap genes in the cell lines may speci cally improve full vector production To test this hypothesis, DHFR gene ampli cation system was employed The DHFR gene driven by the CMV-promoter was cloned into plasmids containing rAAV genome, and stable cell lines were developed using a DHFR de cient cell line, CHO44 The rAAV genomes are ampli ed from several fold to several hundred fold in the cell lines upon treatment with MTX, the chemical that triggers gene ampli cation The ampli ed rAAV genomes remain stable in all cell lines for the passages tested AAV vectors produced were calculated about 800 vector genomes per cell while only about 10% of the cells were transfected with the accessory helper plasmids The AAV vector yields seem corresponding to the copy number of rAAV genomes in the cell lines Studies and attempt to further optimize this encouraging system and eventually develop a gene ampli cation- and mammalian cell-based rAAV production system are being pursued in our lab

674 An Ef cient and Complete Platform for the Production and Use of Lentiviral Vectors

Thomas Quinn,1 Chris Mello,1 Lily Lee,1 Mei Fong,1 Dwayne Bisgrove,1 Michael Haugwitz,1 Hiroaki Sagawa.1

1 Clontech Laboratories, Mountain View, CA.

Lentiviruses are extremely versatile recombinant viral vectors because they can infect, transduce, and sustain expression in almost any mammalian cell type Despite this versatility, many users experience dif culty in achieving timely, robust, and high-ef ciency cell transduction Here we present a complete platform for the ef cient application of lentiviral vectors: from virus production

to post-transduction analysis One common lentiviral issue is low starting titer Previously, we developed a stoichiometrically-optimized packaging mix containing vectors that express high levels of viral packaging proteins from Tet-responsive promoters and an HIV2 LTR Moreover, rapid and ef cient transfection of these plasmids into a high-yield, 293T-based packaging cell line was achieved using

a biodegradable polymer and a short, 4-hour transfection protocol

Taken together, we consistently observed transfection ef ciencies

>99% at 24 hrs and titers >1x107 IFU/ml in 48 hrs To quickly ensure that our transfections yielded high-titer virus, we developed

an HIV-speci c lateral  ow strip, whereby a small amount of crude supernatant (20 µl) is added to the strip and produces a clear signal after ∼2 min Using these strips, we veri ed that our starting titers were high enough (>5x106 IFU/ml) to continue with downstream processes

such as puri cation, concentration, titration, and transduction of

target cells Certain applications, such as in-vivo or high MOI

transductions, bene t from using puri ed, highly concentrated, and low volume preparations Current puri cation methods using anion exchange membranes can potentially shear virions, resulting

in decreased yields and purity (average elution yield of 52%, n=5) Our gentle, anion exchange resin puri cation protocol required only

45 min for 30 ml samples, and resulted in average elution yields

of 74% (n=11) This method also demonstrated improved purity in silver-stained SDS-PAGE gel analyses In addition to puri cation, the common practice of concentration via ultracentrifugation can lead to damaged or aggregated virus and reduced yields due to the extreme forces experienced by the virus As an alternative, we developed a short (∼3 hr), low-speed centrifugation method that demonstrated up to 100-fold concentration of lentiviral stocks In addition, by combining concentration and puri cation of low titer, high volume stocks, we achieved a 19-fold concentration and a 60% yield Because the  nal expression level in a transduced cell depends

on the MOI applied to the cells, and more directly on the number of integrated proviruses, we developed two, vector-speci c, real-time PCR assays to determine both titer and proviral copy number Our 4

hr qRT-PCR assay allowed virus titration and target cell transduction

to be performed in the same day Comparing expression level to copy number, we observed averages of 318 MFI with 1.42 proviral copies in low MOI transductions, and 3566 MFI with 13.4 copies in high MOI transductions Taken together, this platform can decrease lentiviral vector application time to 7 days, reduce repeat experiments, and ensure more consistent and reliable results

675 Development, Scale-Up and Technology Transfer for Rapid, Automated, cGMP Compliant, Large-Volume Suspension Cell Based Process for Lentiviral Vector Production

James Brady,1 Linhong Li,1 Scott R Witting,2 Cornell Allen,1 Rama Shivakumar,1 Aparna Jasti,2 Kenneth G Cornetta,2 Madhusudan V Peshwa.1

1 MaxCyte, Gaithersburg, MD; 2 Vector Production Facility, Indiana University, Indianapolis, IN.

We report here the development, scale-up and technology transfer

to a cGMP facility for a serum-free suspension cell based lentivirus manufacturing process using a rapid, automated, scalable, US FDA Master File supported  ow electroporation system Suspension 293FT cells were chosen for the study Cells were cultured in FreeStyle serum free medium either in shake  asks or in a Wave Bioreactor

No growth difference was found between cell growth methods

A four-plasmid system was used for lentiviral vector production with 293FT suspension cells Virus titer was evaluated following infection of adherent 293T cells over a 2-3d period Viral titer (TU/ mL) was calculated as (% GFP+ cells) * (input cell number)* (Viral supernatant Dilution factor)/(Volume of Viral supernatant) For production of lentiviral vectors, we identi ed an optimal set of processing parameters for loading four DNA component plasmids at two different total DNA concentrations; 400mg/L DNA and 40mg/L DNA At both DNA concentrations, we achieved essentially identical viral titers simply by selecting different sets of process parameters for controlling delivery of electrical energy Viral titers were 9.0 ± 1.4 x107 TU/mL (n=2) and 8.5 ± 1.3 x107 TU/mL (n=4), respectively, with the high and low DNA concentrations Other process parameters that impacted yield and virus titers included incubation post electroporation, conductivity of medium, and cell concentrations Following optimization at small scale, we were able to obtain virus concentration of 1.1 ± 0.4 x108 TU/mL (n=3) at small scale (293 FT cells cultured in shake  ask or in Wave) and 1 ± 0.14 x108 TU/mL (n=2) at large scale (293 FT cells cultured in 2L Wave Bioreactor bags) using reduced DNA concentration of 40mg/ml (0.4mg/ 106 cell) We