597 challenges in the production of large and complex plasmid vectors for gene therapy

597 Challenges in the Production of Large and Complex Plasmid Vectors for Gene Therapy Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy S228[.]

Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy S228

595 Measurement of Viral Titer by Fluorescence

Nanoparticle Tracking Analysis

Andrew Malloy,1 Duncan A Griffi ths,2 Patrick Hole,1 Bob Carr.1

1 NanoSight Ltd., Amesbury, Wiltshire, United Kingdom;

2 NanoSight USA, Costa Mesa, CA.

Measurement of viral titer and sample aggregation is a ubiquitous

requirement in viral vector development Nanoparticle Tracking

Analysis (NTA) is a new methodology which provides total viral

titer in minutes and real time measurement of sample aggregation

The ability to measure these parameters at key points throughout the

downstream purifi cation process allows manufacturers to monitor

and optimize the sample purifi cation The technique images viruses

individually in liquid suspension and then calculates size from their

Brownian motion on a virus-by-virus basis By individually counting

and sizing the viruses, a high resolution number vs size distribution is

generated which relates the number of virus monomer to aggregates

Total virus count or concentration is simultaneously derived from this

measurement Fluorescence based measurements allow speciation of

appropriately labeled sub-populations within the sample Fluorescent

labeling of the virus capsids, envelope, or DNA allows distinction of

virus from cell debris making the technique suitable for working in

crude harvest materials Operating under light scatter the technique

is inherently non-specifi c and hence suited to working in purifi ed

samples As all particles, virus or otherwise, are measured, this can be

combined with the fl uorescence measurements to provide a measure

of contaminant or empty capsid concentrations The technique is

designed to work alongside traditional technologies such as infectivity

assays, as these assays provide valuable, yet limited information

Infectivity assays have no ability to pick up aggregation within a

sample and do not give a measure of total viruses within a sample

When this data set is merged with the NTA data, the user can monitor

infective vs non-infective viruses vs sample aggregation to better

understand the quality of a viral preparation

596 Where Human Gene Transfer Is Illegal:

Local Regulation of rDNA Clinical Research

Jan P Vleck,1 Gary M Johnson,2 Ethan Mascoop.3

1 IBC Services, Western Institutional Review Board, Olympia, WA;

2 MetroWest Medical Center, Framingham, MA; 3 Framingham

Board of Health, Framingham, MA.

“The use of humans as experimental subjects in recombinant

DNA research, as defi ned and regulated by the NIH Guidelines,

shall not be permitted in the Town of Framingham.” [Board of

Health, Rules and Regulations Relative to the Use of Recombinant

DNA Technology within the Town of Framingham]1 A local regulation

outlawing the use of human subjects in recombinant DNA (rDNA)

research was discovered during preparation for a

commercially-sponsored, multicenter, Phase II human gene transfer trial at a hospital

in Framingham, Massachusetts Penalties for violation including fi nes

and closure of the “laboratory” The hospital succeeded in obtaining

a variance allowing the research

Selected Requirements of the Framingham BOH Regulation

conform to NIH Guidelines intestinal fl ora surveillance

BOH-approved procedure manual investigate and report all worker illness

emergency response plan ban on P3, P4 research

at least 2 BOH-appointed IBC members ban on use of human subjects

IBC minutes to BOH institutional registration with BOH

local screening for purity and antibiotic

resistance fi ne $200/day and lab closure

Local Context A controversial 1976 Harvard University plan

for a P3 research laboratory in downtown Cambridge prompted

various local actions to regulate use of rDNA In Framingham, the

Town Board of Health is charged with “registering recombinant

DNA technologies.”2 MetroWest Medical Center (MWMC) is an

independent regional health care system serving the Framingham

area Opening the trial On September 14, 2010 MWMC registered an

IBC with NIH OBA in preparation for opening its fi rst gene transfer trial IBC review was scheduled for October 19 On October 7, a local ban on human gene transfer, probably from the early 1980s, was discovered MWMC considered several options Abandon the trial This was not acceptable Move the trial Logistical and public relations considerations made it untenable to relocate to a MWMC hospital in neighboring Natick Modify the regulatory status This could both address public health concerns, and increase the feasibility

of future HGT research in Framingham This option was chosen On October 8, the IBC roster was re-registered to add two Town residents unaffi liated with MWMC (one the Director of Public Health) MWMC submitted background safety information to the BOH and requested

a permanent variance allowing FDA regulated, industry sponsored rDNA clinical trials On October 28, the BOH approved a single-study variance as a test of procedural competence and safety Legal notice

of the BOH decision and amendments to the regulation was printed

in the local paper The BOH fi led its actions with the Massachusetts Department of Environmental Protection On November 10, the IBC approved the research The local newspaper ran two stories.3,4 The

fi rst screening visit occurred January 11, 2011 Discussion: A local regulation outlawing human gene transfer research caused a 3-week delay in IBC approval for this regional community hospital site in a multi-center trial The strategy of seeking a regulatory variance, going public in the local newspaper, and expanding public participation in the IBC was successful

1 http://www.framinghamma.gov/DocumentView.aspx?DID=2812, accessed 01-11-2011[Home/Government/Departments/Board of Health/Rules and Regulations/Recombinant DNA Technologies Regulation]

2 http://www.framinghamma.gov/index.aspx?NID=852, accessed 01-11-2011 [Home/Government/Departments/Board of Selectmen/ Selectmen Appointed Committees/Board of Health]

3 Morton, M MetroWest Medical seeks update to Framingham’s DNA regulation The MetroWest Daily News http://www metrowestdailynews.com/lifestyle/health/x370073169/MetroWest-Medical-seeks-update-to-Framinghams-DNA-regulation, posted 10-29-2010, accessed 01-11-2011

4 Morton, M MetroWest Medical Center approved to for [sic] HPV treatment trial The MetroWest Daily News http://www metrowestdailynews.com/lifestyle/health/x600431471/MetroWest-Medical-Center-approved-to-for-HPV-treatment-trial, posted

11-14-2010, accessed 01-11-2011

597 Challenges in the Production of Large and Complex Plasmid Vectors for Gene Therapy

Ying Cai,1 Stephen Rodriguez,1 Luke Clifford,1 Henry L Hebel.1

1 VGXI, Inc, The Woodlands, TX.

Regulatable gene therapy appears as a promising approach by adjusting or switching on/off gene expression on demand However, along with advantages of safety and fl exibility, the size and complexity

of the vector are increased to accommodate various control elements

A number of obstacles arise during production of such plasmids at pre-clinical or clinical grade, such as: low yield, instability, shear-sensitive, and abundance of impurities To address these challenges, VGXI devised process development programs for each stage of plasmid production Extensive screening and bioreactor simulation ensured the selection of a high quality and high yield clone for seed banks Large plasmids with aberrant sequences typically have high recombination ratios, and the quality of the desired plasmid form

is associated with growth temperature We have found the normal growth temperature of 37°C led to an unacceptable high level of recombinants Reducing grow temperature improved product quality, but resulted in several fold reduction of initially low copy number plasmids To accommodate both quality and yield, a novel fed-batch strategy was developed, which not only minimized undesirable forms

Molecular Therapy Volume 19, Supplement 1, May 2011

CLINICAL GENE & CELL THERAPY ORAL ABSTRACT SESSION

but also increased yield titer up to 4-fold Downstream processing also

faced unusual barriers because large plasmids share similar physical/

chemical characteristics contaminating genomic DNA Alkaline lysis

is of particular concern as large plasmids are ineffi cient at renaturing

Special attention to process shear is required to maintain structural

integrity We have optimized downstream process conditions at every

step For a plasmid of size greater than 12 kb, the product demonstrated

high purity suitable for pre-clinical or clinical applications

Clinical Gene & Cell Therapy Oral Abstract Session

598 A Gene Therapy Approach to HIV: Adoptive

Transfer of Zinc Finger Nuclease (ZFN) Modifi ed

Autologous CD4 T-Cells to Aviremic HIV-Infected

Subjects with Suboptimal CD4 Counts (200-500

Cells/mm3)

Shelley Wang,1 Jay Lalezari,2 Ronald Mitsuyasu,3 Steven Deeks,4

Winson Tang,1 Gary Lee,1 Michael Holmes,1 Phillip Gregory,1

Marty Giedlin,1 Dale Ando.1

1 Sangamo Biosciences, Richmond, CA; 2 Quest Clin Research, San

Francisco, CA; 3 UCLA, Los Angeles, CA; 4 UCSF, San Francisco,

CA.

Background: A signifi cant number of HIV+ patients on HAART

are aviremic but continue to have CD4+ T-cells <500 cells/mm3

Previous attempts to use adoptive transfer to protect CD4+ T-cells

from HIV infection have shown limited cell persistence ZFN

mediated disruption of CCR5 in CD4+ T-cells and selective expansion

has been shown to protect against R5 HIV infection in both in vitro and

in vivo T-cell and stem cell models This study assesses the following

effects of a single infusion of autologous CCR5-disrupted CD4+

T-cells administered intravenously to HAART-treated aviremic HIV

subjects: 1) safety and tolerability, 2) cell persistence, 3) increases

in CD4+ cell count, and 4) homing to gut mucosa Methods: Nine

aviremic HIV infected subjects with CD4+ counts between 200 and

500 cells/mm3 were enrolled, three each at dose levels of 1X1010,

2X1010 and 3X1010 total cells Autologous CCR5 disrupted CD4+

T-cells were successfully expanded ex vivo with a mean CCR5

disruption effi ciency of 26% (range 14-36%) After infusion, subjects

were followed weekly for one month and then monthly for 11 months

Results: The median follow-up to date for cohort 1 is 9 months

(range 7-11) CCR5-disrupted CD4+ T-cell infusions were safe and

well tolerated with only mild and reversible infusion related adverse

effects such as fever and chills CD4 T-cell counts increased at all time

points post infusion in all subjects The average CD4+ T-cell counts

at Day 28 increased by 155 cells/mm3 (range 86-211) The number of

CCR5-disrupted T-cells in the peripheral blood as measured by PCR

was 3.9-, 0.3- and 4.6-fold of the predicted number (2.5% total CD4

in 4.7L of blood) at Day 14 and persisted for the duration of

follow-up (7 to 11 months) The percentage of CCR5 disrfollow-upted CD4 cells

detected in the peripheral blood was as high as 19% in one subject

at Day 28 CCR5 disrupted cells were detected in the rectal mucosa

of all cohort 1 subjects at Day 14 and at 3 months Conclusions:

CCR5 disrupted CD4+ T-cells can be consistently manufactured

from patients with suboptimal CD4+ counts to generate amounts

greater than the intended 10-30 billion cell dose Reinfusion to

HIV-infected subjects is safe and well tolerated with disrupted cells being

detected at frequencies up to 4.6-fold higher than the predicted input

14 days after infusion This level of gene marking is approximately

1-log greater than has been observed previously in adoptive transfer

studies with co-stimulated CD4+ T cells Improvements in CD4+

T-cell counts were seen in all 3 subjects as was homing of these

modifi ed cells to the gut mucosa, suggesting that the modifi ed CD4+

T-cells may distribute normally These preliminary data suggest that

the CCR5 disrupted CD4 T cells persist at signifi cant levels post infusion and can bolster CD4 cell counts even in HIV patients with undetectable viral load

599 Clinical Trial Results: Intralesional Injection

of a Tumor Selective Oncolytic Vaccinia Virus

Herbert J Zeh,1 Mark O’Malley,1 Heather Jones,1 David H Kirn,2

Moon Anne,2 Hwang H Tae,3 David L Bartlett.1

1 Surgery, University of Pittsburgh, Pittsburgh, PA; 2 Jennerex Biotherapeutics, Inc., San Francisco, CA; 3 Dong-A University, Busan, Korea.

Introduction: The WR strain of vaccinia virus (VV) has numerous

potential advantages as an oncolytic virus We have created a tumor selective mutant of VV (vvDD) by deleting the thymidine kinase (TK) gene and the vaccinia growth factor (VGF) gene These gene products are non-essential in tumor cells with E2F and/or EGFR pathway activation mutations (including ras), but essential for replication in normal, non-dividing cells After extensive pre-clinical work, we embarked on a phase I trial of vvDD as an intralesional injection in

patients with accessible tumors Methods: Patients with metastatic

breast cancer (n=4), colorectal cancer (n=10), and pancreatic cancer (n=2) were treated with a single percutaneous injection of vvDD A standard phase I dose escalation was performed with a starting dose

of 3X107 plaque forming units (pfu), and a maximum feasible dose

of 3X109 pfu Up to 3 lesions were injected Patients were observed

in the hospital for 24 hours, then followed closely with serial samples for viral shedding analysis and analysis of response Cutaneous tumors allowed the direct observation of the response and biopsies for viral recovery, while CT scans were used for internal lesions Patients were stratifi ed for prior vaccinia exposure, but only one patient had not

been previously vaccinated Results: There were no dose limiting

toxicities Grade 3 toxicities possibly related to the treatment, included pain after injection This was predominately abdominal pain after deep injection of hepatic metastases Grade 1 and 2 adverse events included fever, chills, swelling and erythema at the injection site, diffuse rash (not systemic vaccinia), and thrombocytopenia We did not see evidence of viral shedding by vaccinia titers 4 patients underwent biopsies of their tumors on day 8, and 2 patients had recoverable virus These 2 patients also had evidence of viral spread from the injected lesions to other metastatic deposits 2 patients had complete resolution of their injected lesions, and 1 patient had evidence of a distant lesion responding The virus did not spread to normal skin

Conclusions: We have demonstrated the safety of vvDD as a single

injection into tumors, with evidence of replication, tumor response, and recoverable virus in distant tumors The vaccinia replication was specifi c for tumor, with complete sparing of the normal skin Future plans include systemic delivery of this tumor selective virus

600 Safety and Effi cacy of Re-Administration of AAV2.hRPE65v2 in Subjects with Leber Congenital Blindness Due to RPE65 Mutations

Jean Bennett,1,2 Albert M Maguire,1,2 Federico Mingozzi,2 Eric A Pierce,1,2 Daniel C Chung,1,2 Jeannette Bennicelli,1 Junwei Sun,2

J Fraser Wright,1,2 Kathleen Marshall,2 Jennifer M Wellman,2

Katherine A High.1,2

1 Ophthalmology, University of Pennsylvania, Philadelphia, PA;

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

Gene therapy has the potential to reverse disease or prevent further deterioration of vision in patients with incurable inherited retinal degeneration We and others previously demonstrated that adeno-associated virus (AAV)-mediated delivery of RPE65 via subretinal injection results in improved vision/retinal function in animal models

of and in humans with Leber Congenital Amaurosis due to RPE65