411 a helper dependent adenovirus epstein barr virus hybrid system that stably transforms cells in vitro with high efficiency

411 A Helper Dependent Adenovirus Epstein Barr Virus Hybrid System that Stably Transforms Cells in Vitro with High Efficiency Molecular Therapy �������� ��� ���� ���������������� �������� ���� ������©[.]

Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts

Copyright © The American Society of Gene Therapy

S162

VECTOR GENOME BIOLOGY

positive for vector sequences in the period immediately following

vector injection Adult animals (n=27) were injected at doses ranging

from 1 x 10E11 to 1 x 10E13 vg/kg and semen samples were collected

weekly DNA obtained from total semen, motile, and non-motile

sperm fractions were assayed by the same sensitive PCR developed

for the clinical study We found that rabbits uniformly showed a

positive signal in serum at 7 days post injection Moreover, there

was a dose-dependent increase in the likelihood of finding vector

sequences in semen DNA Thus, animals injected at 1 x 10E11 vg/

kg were initially positive but cleared by week 4 following injection

However, animals injected with a dose of 1 x 10E12 vg/kg or 1 x

10E13 vg/kg required 8- 13 weeks to clear Semen fractions enriched

for motile sperm were transiently positive and the clearance was

faster than for total semen specimens These data suggest that

clearance of vector sequences from semen DNA eventually occurs

and that the kinetics of clearing is dose-dependent Findings from

the clinical study demonstrated PCR positivity in the semen samples

from all subjects enrolled (AAV vector dose ranging from 8 x 10E10

to 2 x 10E12 vg/kg) when samples collected at day 7 following

injection were assayed To address the duration of shedding of

infectious AAV particles into the semen, we carried out an AAV

infectivity assay using serial semen samples collected from injected

rabbits The results demonstrated that even at the earliest time point

assayed (day 7) no infectious AAV particles were detected in the

semen as was also the case for semen samples collected at later time

points Animals followed for over a year (more than 8

spermatogenesis cycles) do not reveal any subsequent recurrence of

vector sequences in the semen, which suggests that no early

spermatogonial precursors were affected by vector In addition, for

subjects below 40 years of age, the period of vector shedding was

4-6 weeks, considerably shorther than that observed for of older

subjects (8-12 weeks) Together these results suggest that the risk

of persistent PCR positivity of semen DNA for vector sequences is

low, and that AAV infectious particles are not present even in samples

collected at early time points The data also suggest that the duration

of vector shedding may be shorter for younger patients

Dr Couto, Dr Kay, Ms Chew, Dr Zhen and Dr Sommer have

equity at Avigen, Inc

NOVEL AD VECTORS - AD RECEPTORS

410 Determinants of and Pharmacologic

Modulation of Acute Toxicity Associated with

Systemic Administration of First Generation and

Helper-Dependent Adenoviral Vectors

Gabriele Toietta,1 Viraj P Mane,1 Lucio Pastore,2 Milton J

Finegold,3 Philip Ng,1 Arthur L Beaudet,1 Brendan Lee.1,4

1 Molecular and Human Genetics, Baylor College of Medicine,

Houston, TX, United States; 2 Dipartimento di Biochimica e

Biotecnologie Mediche and CEINGE-Biotecnologie Avanzate,

Universita’ degli Studi di Napoli Federico II, Naples, Italy;

3 Pathology, Baylor College of Medicine, Houston, TX, United

States; 4 Howard Hughes Medical Institute, Baylor College of

Medicine, Houston, TX, United States.

Understanding how systemic administration of adenoviral (Ad)

vectors elicits an acute host response is critical for human clinical

gene therapy Toxicity associated with systemic Ad administration

is the result of a multi-phasic process Early, acute toxicity is related

to viral interaction with target cells and/or consequent vectors entry

with viral uncoating This process occurs rapidly within minutes to

hours of vector administration The activation of cytokines and

thrombocytopenia associated with acute toxicity may result, at high

doses, in disseminated intravascular coagulation and multi-organ

damage Intermediate toxicity, occurring from hours to days after

injection, is less well defined and likely involves de novo cellular

transcription and amplification of cytokine release and the systemic inflammatory response Several days after Ad administration chronic toxicity may arise It is primarily mediated by the host adaptive

immune response to de novo production of antigens In early

generation Ad vectors, the primary antigens derive from viral gene expression, though potential neo-antigenicity of the therapeutic transgene may also contribute Indicators of vector-associated toxicity include elevation of cytokine levels, including IL-6, and increase in plasma levels of liver transaminases and reduction in circulating platelet numbers We evaluated these parameters to compare early and intermediate toxicity associated with systemic administration

of equal doses of first generation (FG-Ad) and helper-dependent (HD-Ad) vectors containing the same expression cassette in C57BL/ 6J mice We confirmed that FG-Ad and HD-Ad have a different effect on chronic toxicity but induce a similar acute toxicity profile

in the first 12 hours after systemic administration In order to further elucidate the genetic determinants of this host response we administered Ad vectors into mice compromised in different immune signaling pathways Our data suggest an involvement of an early

proteins as a possible event leading to the dose-dependent thrombocytopenia associated with adenoviral vector administration Furthermore we exclude a significant contribution of interferon-γ and perforin-mediated pathways in acute and intermediate toxicity Finally we determine that mast cell depletion does not improve platelet counts measured 72 hours after Ad vectors injection

reduced early toxicity associated with vector administration as shown

by decreases in IL-6 levels and thrombocytopenia, without concurrent effect on efficiency of transduction or IL-12 levels Therefore, the use of HD-Ad vectors in combination with

index of human gene therapy trials

411 A Helper Dependent Adenovirus-Epstein Barr Virus Hybrid System that Stably Transforms Cells in Vitro with High Efficiency

Oliver Dorigo,1 Jose S Gil,1 Sean Gallaher,1 Brent Tan,1 Arnold J Berk.1

1 Molecular Biology, UCLA, Los Angeles, CA, United States.

Epstein Barr Virus (EBV) episomes are stably maintained in proliferating cells due to Epstein Barr Nuclear Antigen-1 (EBNA-1) protein mediated segregation and replication once per cell division

We have previously reported the delivery of an EBV episome using

an adenovirus-EBV hybrid system (Tan et al, J Virol, 73(9), p.7582) This system utilizes Cre/loxP mediated recombination of the linear adenoviral genome into a circular episome that stably transforms canine D17 cells However, maximum transformation efficiency required doses of the E1-deleted adenoviral vectors that caused significant toxicity to infected cells

In the present study, we have utilized a binary helper dependent adenoviral (HDA) vector system to deliver an EBV episome using Cre/loxP recombination Our approach included the design of a HDA for the expression of Cre (HDA.Cre) and a second HDA for the delivery of the EBV episome (HDA.PAC and HDA.CFP) All vectors contain an expression cassette for yellow fluorescent protein for titration The sequences for the EBV episome - including the EBV family of repeats, an EBNA-1 expression cassette, the transgenes for puromycin acetyltransferase (PAC) or cyan fluorescent protein (CFP), as well as a 19kb human DNA sequence for replication - were flanked by loxP sites Upon co-infection of canine D17 and human HeLa cells with HDA vectors in vitro, the loxP vectors underwent Cre induced recombination and generation

of an EBV episome as assayed by Southern Blot analysis

Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts

NOVEL AD VECTORS - AD RECEPTORS Using a colony-forming assay in puromycin selection medium,

we demonstrated stable transformation of up to 61% of D17 cells

after co-infection with HDA.Cre and HDA.PAC Furthermore, cell

viability at the MOI necessary to achieve this transformation

efficiency was significantly better compared to the E1-deleted system

(66.2% vs 5.4%) Due to reduced cytotoxicity, the HDA-EBV

system was found to be significantly more efficient compared to the

E1-deleted system Stable transformation was also achieved in 16%

of co-infected HeLa cells without significant cytotoxicity

To further optimize the binary HDA-EBV system, we used CFP

as marker for recombination Due to different excitation and emission

spectra, CFP as expressed on the episome can be distinguished for

YFP on the linear vector using fluorescence microscopy

Interestingly, variations of the MOI for HDA.Cre and HDA.CFP

resulted in significantly different recombination efficiencies calculated

as percentage of CFP positive cells We found that co-infection of

HDA.Cre at MOI=10 (titered by YFP transduced units) and

HDA.CFP at MOI=30 resulted in 40-50% CFP positive cells after

7 days A further increase of the MOI for HDA.Cre surprisingly

resulted in a reduction of recombination efficiency, possibly due to

competition between the two vectors for cell entry In contrast, an

increase of the MOI for HDA.CFP resulted in a slightly greater

percentage of recombination but also a decrease in cell viability

In summary, we have constructed a HDA-EBV hybrid system

capable of stably transforming mammalian cells in vitro Due to

significantly reduced cytotoxicity, this system was found to be

superior to the previously reported E1 deleted adenovirus mediated

EBV delivery system

412 Z Domain-Modified First-Generation and

High-Capacity Adenoviral Vectors for

Antibody-Mediated Targeted Gene Transfer

Christoph Volpers,1 Volker Biermann,1 Christian Thirion,2

Stefanie Hussmann,3 Helmut Kewes,1 Andreas Herrmann,3 Hanns

Lochmueller,2 Stefan Kochanek.1

1 Center for Molecular Medicine (ZMMK), University of Cologne,

Cologne, Germany; 2 Gene Center, University of Munich, Munich,

Germany; 3 Cardion AG, Erkrath, Germany.

In order to target adenoviral (Ad) vectors to cell types lacking or

expressing low levels of the Ad receptor, target-specific peptide

ligands have been genetically incorporated into the capsid, fiber

proteins from Ad serotypes with different tropism have been

inserted, and bispecific recombinant or chemically engineered adaptor

molecules have been used However, small, high-affinity binding

peptide ligands suitable for capsid incorporation are available only

for few cell surface molecules, and all these strategies have to be

specifically tailored for each respective target receptor or cell type

Therefore, we have developed an efficient and highly versatile new

strategy allowing the direct use of monoclonal antibodies against

cell surface antigens for targeting of Ad vectors A full-length

Z-domain (Z59; 59 amino acids) or a shorter version of it (Z33; 33

amino acids), IgG-binding domains derived from staphylococcal

protein A, were inserted into the Ad fiber protein Both modified

fiber proteins retained the ability to assemble into trimers, they

bound IgG with high affinity as demonstrated by ELISA and surface

plasmon resonance measurements, and they were efficiently

incorporated into vector particles Pull-down experiments with

IgG-Sepharose and competition ELISA confirmed IgG-binding activity

of the Z-modified vectors By preincubation with different

concentrations of a monoclonal antibody directed against epidermal

growth factor receptor (anti-EGFR), transduction efficiencies of

the Z33- and the Z59-modified first-generation vectors in

EGFR-overexpressing A431 carcinoma cells were increased in an antibody

dose-dependent manner by up to 18-fold and 50-fold, respectively

The increase in gene transfer levels was receptor-specific and could

be competitively blocked by protein A and by recombinant Z-modified knob protein, but not by wild-type knob By use of a Z59-modified helper virus, a high-capacity or ‘gutless’ vector was produced which could be targeted to EGFR in the same way Pretreatment of the Z33-modified first-generation vector with a monoclonal antibody recognizing integrin alpha7 considerably increased transduction of primary human smooth muscle and skeletal muscle cells In primary human myotubes, which are particularly refractory against Ad infection, preincubation of this vector with antibodies (at 40 microgram/ml) specific for the cell surface molecules integrin alpha7 or NCAM enhanced transduction efficiency by 68-fold and 77-68-fold, respectively, as assessed by quantitative beta-Gal reporter gene assay The Ad vectors described hold great potential for targeting to a wide variety of cell types by simply exchanging the monoclonal antibody

413 Analysis of High-Capacity Adenovectors Expressing PEDF for Ocular Gene Therapy

Joseph T Bruder,1 Ping Chen,1 Kurt Kroeger,1 Alena Lizonova,1

C R King.1

1 New Products Research, GenVec Inc., Gaithersburg, MD, United States.

Wet age-related macular degeneration (AMD) and diabetic retinopathy are the most common causes of blindness in the developed world Both diseases are caused by the abnormal growth

of blood vessels in the eye Current treatments that destroy or inhibit the growth of existing blood vessels can delay vision loss but

do not affect the underlying disease and neovascularization is a recurring problem Pigment epithelium-derived factor (PEDF) is a potent anti-angiogenic and neuroprotective protein that is naturally produced in the eye When delivered to the eye via an adenovirus vector, PEDF can block growth of new blood vessels and trigger the regression of established abnormal vessels in animal models for AMD and diabetic retinopathy Two characteristics of AdPEDF that have been observed in animal models are the potential for vector induced inflammation and transient PEDF expression High-capacity (HC) adenovectors are characterized by the absence of viral coding sequence and are also referred to as helper-dependent, gutless or gutted vectors These vectors have been shown to be the least immunogenic, least toxic, and most persistent adenovectors to date Due to the absence

of adenovirus genes, HC adenovectors offer the potential for persistent transgene expression and may greatly expand the therapeutic repertoire for adenovirus gene therapy vectors in the eye and other tissues We have built a functional system for producing gutless vectors that includes a cell line that expresses the FLPe recombinase (293FLPe17); a helper virus that has its packaging signal flanked by frt recombination sites; and HC vectors that express green fluorescent protein (GFP) and PEDF We observed efficient (>95% ) packaging signal excision at both 24 and 48hours after infection of 293FLPe17 cells with the frt helper virus In addition, helper virus yields were reduced by more than 500-fold in 293FLPe17 relative to 293 cells These results indicate that the FLPe system is functional We have used both this FLPe/frt and the Cre/Lox system

to grow a HC vector that expresses the PEDF gene from the CMV promoter HC-vector preparations containing less than 1% helper contamination were obtained following CsCl purification We are currently investigating the inflammatory response to these vectors and the persistence of PEDF expression following delivery to the mouse eye

I work for and hold stock in GenVec