14 immunosuppression with cyclophosphamide significantly prolongs high level expression of IDUA mediated by the sleeping beauty transposon system

14 Immunosuppression with Cyclophosphamide Significantly Prolongs High Level Expression of IDUA Mediated by the Sleeping Beauty Transposon System Molecular Therapy Volume 19, Supplement 1, May 2011 Co[.]

Molecular Therapy Volume 19, Supplement 1, May 2011

expression was silenced as with the standard plasmid vector These

results suggested that spacer DNA regardless of its source >1kb

caused the minicircle constructs to be silenced even in the absence of

plasmid BB To further establish this idea, 500bp of plasmid BB was

used as spacer to make new constructs These constructs also showed

persistent expression suggesting the size of the non-transcribed insert

and not the source of the spacer DNA was the critical factor for DNA

silencing To better understand the mechanism of plasmid silencing,

we attempted to determine if leaky transcriptional termination across

the spacer sequences was an important parameter for silencing To do

this, we added a robust transcriptional terminator between the poly

A site and DNA spacer Even with an additional terminator, only

spacers of > 1kb silenced the transgene Our data suggest that the

distance between the poly A site and promoter region infl uences DNA

silencing These results have important implications for designing

DNA plasmid vectors for gene therapy

Quantitative Method for Assessing Plasmid

Nuclear Import

Holly M Reynolds,1 David A Dean.1

1 Pediatrics, University of Rochester, Rochester, NY.

One area of interest in gene therapy is designing strategies that

can allow DNA to be delivered only to a specifi c cell type or tissue

Historically, these strategies have included: application of the agent

directly to desired tissue, exploitation of receptor-ligand interactions,

and using cell-specifi c promoters to drive transcription Our lab has

developed a novel and robust method for cell-specifi c delivery that

allows for nuclear import in non-dividing cells by a sequence specifi c

process that requires the cytoplasmic binding of transcription factors,

these factors provide transport across the nuclear membrane Namely,

this exploits the fact that promoter sequences bind transciption factors

and in some cases, when the Nuclear Localization Signal is oriented

in the correct way, allow for taxiing across the nuclear membrane To

date, we have discovered DNA sequences that provide cell-specifi c

import in smooth muscle cells, osteoblasts, endothelial cells and

alveolar type 2 epithelial cells Identifying such plasmid sequences,

however, is a tedious and time-consuming task, requiring cytoplasmic

microinjection of individual plasmids containing potential sequences

into hundreds to thousands of individual cells and determining

nuclear localization of the plasmids by in situ hybridization and/

or fl uorescence microscopy We have developed a simpler,

higher-throughput assay that allows us to screen a large number of potential

sequences for cell-specifi c nuclear entry in a large panel of cell types

Using a 96-well electroporator with optimized fi eld strengths, we

can induce cell uptake of pools of fl uorescently-labeled plasmids

containing various DNA sequences After incubation, these cells are

fi xed and run, in a 96-well format, on an AMNIS Imagestream with

a nuclear stain This instrument is essentially a fl ow cytometer that

images each cell in up to 8 fl uorescent channels with a 40X or 60X

objective as it moves through the stream The instrument is capable

of collecting data from up to 5000 cells per minute and images can

then be analyzed with software to assess and quantify subcellular

localization of the plasmids Using this approach, we have shown

that plasmids carrying the ubiquitously active SV40 DNA nuclear

targeting sequence localize to the nuclei of multiple cell types while

isogenic plasmids lacking this sequence remain in the cytoplasm

unless incubation times are greatly increased to allow for cell division

(and concomitant breakdown of the nuclear envelope) This approach

should greatly enhance our ability to screen DNA sequence libraries

in a wide variety of cell types to identify nuclear localizing DNA

sequences for cell-specifi c gene therapy

in Mouse Liver Via the Novel hAT Transposon

TcBuster

Lauren E Woodard,1 Aparna Kaja,1 Robert H Hice,2 Peter W Atkinson,2 Nancy L Craig,3 Matthew H Wilson.1

1 Department of Medicine, Division of Nephrology, Baylor College

of Medicine, Houston, TX; 2 Department of Entomology &

Institute for Integrative Genome Biology, University of California, Riverside, Riverside, CA; 3 Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, MD.

TcBuster is a recently discovered mobile DNA element from the hAT superfamily Colony count assays in HEK 293 cells using

varying amounts of transposon and transposase indicate that maximal transposition occurred using 500 ng of pTCBNeo and 100 ng of

pCMV-TCB Out of 48 TcBuster integration sites sequenced from

these cells, 62.5% were in genes and 4.2% were in exons, suggesting

a preference for transcriptionally active areas Comparing different transposons by colony count assay in HEK 293 cells, the transposition

of TcBuster was approximately equivalent to hAT family member Tol2 and about half that of piggyBac, depending on the ratio of transposon

to transposase plasmids When an equal amount of transposase and transposon plasmids were used, all three systems were approximately equal However, when an excess of transposase (200 ng of transposon and 800 ng of transposase plasmids) or an excess of transposon (900

ng of transposon and 100 ng of transposase plasmids) were provided,

piggyBac outperformed both hAT systems, indicating the signifi cant

fl exibility of the piggyBac system As for the hAT systems, when the amount of transposase plasmid was higher, the Tol2 system was superior to TcBuster, whereas when the amount of transposase plasmid was low, the TcBuster system was superior to Tol2 After transfection with a construct carrying an HA-tagged version of the TcBuster

transposase, staining revealed localization to unique intranuclear rodlets We hypothesize that the rodlets may serve a protective role, limiting free transposase and thus DNA damage In support of this hypothesis, as the amount of pCMV-HA-TCB is increased beyond

100 ng, the number of rodlets per nuclei increased exponentially as

the number of colonies formed decreased To compare TcBuster with piggyBac for integration into mammalian chromosomes in vivo, adult

female FVB mice were given hydrodynamic injections containing luciferase transposon plasmids with or without the plasmid encoding the transposase Mice were imaged using the IVIS system to determine the levels of liver-specifi c luciferase expression at timepoints out to

24 weeks At the later timepoints, the normalized luciferase values

for groups given either TcBuster or piggyBac transposase were

both statistically signifi cantly higher than groups given transposon alone (p<0.0005 and p<0.01, respectively, by two-tailed t-test) An

equivalent t-test comparingTcBuster with piggyBac revealed that

they were not signifi cantly different from one another Therefore, we

conclude that TcBuster is an exciting new transposon that is highly capable of providing long-term gene expression in vivo.

Cyclophosphamide Signifi cantly Prolongs High-Level Expression of IDUA Mediated by the

Sleeping Beauty Transposon System

Elena L Aronovich,1 Jason B Bell,1 Kelly M Podetz-Pedersen,1

Brenda L Koniar,2 R Scott McIvor,1 Perry B Hackett.1

1 Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN; 2 Research Animal Resources, University of Minnesota, Minneapolis, MN.

Mucopolysaccharidosis type I (MPS I) is a systemic disease caused

by defi ciency of the lysosomal enzyme α-L-iduronidase (IDUA) The goal of gene therapy for MPS I is to achieve sustained expression

Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy S6

of an IDUA transgene We have reported the effectiveness of the

Sleeping Beauty (SB) transposon system for gene therapy of MPS

I in adult immunodefi cient mice However, in immunocompetent

mice IDUA expression is curtailed mainly by immune responses

to the transgenic IDUA polypeptide Although more than 99% of

transgene expression comes from the liver following hydrodynamic

delivery, restoration of defi cient enzyme activity in other organs

can be achieved through metabolic cross-correction whereby IDUA

enters the circulation and is distributed to other tissues The IDUA

gene must therefore be regulated by a strong promoter that will

drive high-level expression, i.e ≥ 100-fold the wild-type (WT) level

We have previously shown that a strong liver-specifi c promoter

ApoEhAAT has several advantages over using the strong ubiquitously

expressed CAGGS promoter to drive IDUA including: 1) increased

IDUA expression level at 5-6 weeks post-infusion and 2) absence

of a gender bias that we characterized using the CAGGS promoter

for expression in MPS I mice We hypothesized that a combination

of immunosuppression and liver-specifi c expression would increase

the levels of IDUA maintained in immunocompetent MPS I mice

We hydrodynamically injected 5 µg pT2/ApoEhAAT-IDUA SB

transposons into immunocompetent C57BL/6 mice with or without

pCMV-SB100 transposase (+ or -SB) Preliminary comparison of

transposition effi ciency by real-time qPCR excision assay showed that

SB100 was at least 30 times more effective than SB11 in mouse liver

For long-term studies, mice (n=10 mice /cohort) were treated with

or without cyclophosphamide (50 mg/kg i.p on -1 day, -5 h, +1 day,

and +2 days with respect to hydrodynamic injection) IDUA activity

in plasma was 200-300-fold greater than WT in all mice 1-day

post-injection (p.i.) Without cyclophosphamide, plasma IDUA declined

slightly over the fi rst two weeks but thereafter deteriorated in most

mice (+ and -SB) to background levels by six weeks p.i In a few mice

that were not immunosuppressed, IDUA activity remained consistent

for 6 - 12 weeks before declining to background over a period of 2

weeks In contrast, IDUA expression levels were maintained in all

except 5 of the 19 cyclophosphamide-treated mice and at 12 weeks

p.i IDUA expression was 217±159-fold WT (+SB) and 75±60 (-SB)

Thus, the combination of transient immunosuppression and a

liver-specifi c promoter resulted in sustained IDUA expression at levels that,

with SB100, were therapeutically relevant for treating MPS I

Mediated Gene Transfer in Human Cells

Sunandan Saha,1,2 Aparna Kaja,1 Cliona M Rooney,2,3 Matthew H

Wilson.1,2

1 Department of Medicine- Nephrology Divison, Baylor College of

Medicine, Houston, TX; 2 Program in Translational Biology and

Molecular Medicine, Baylor College of Medicine, Houston, TX;

3 Department of Pediatrics, Baylor College of Medicine, Houston,

TX.

PiggyBac has been successfully used for modifi cation of primary

human cells and cell lines with transgene(s) of interest and holds

promise as an effective non-viral gene delivery method Currently,

limited information exists about the safety of the piggyBac system for

the modifi cation and generation of clinical grade human cells In this

study we began to evaluate the safety of piggyBac for modifi cation of

human cells PiggyBac works through a “cut and paste” mechanism

thereby delivering a transgene of interest fl anked by inverted repeat

sequences into the genome We found that piggyBac leads to stable

transgene integration and transposase expression is undetectable after

7 days in a mixed population of human cells Although there are no

sequences in the human genome with complete similarity to the 17bp

terminal repeat sequence (TRS) of the piggyBac transposon inverted

repeats (IR), there are sequences with 16, 15 or 14 bp similarities all

of which end in the tetranucleotide TTAA required for transposition

We subsequently tested the ability of piggyBac to mobilize these

genomic elements The potential of these sequences mimicking the TRS was assessed by replacing the TRS of the 5’Inverted repeat with these genomic sequences and looking at transposition effi ciency using a colony count assay None of the 14 tested sequences were able to effectively act as a terminal repeat sequence Nor did they mediate excision of transposons in presence of the transposase To assess the safety of delivering the transposase and transposon from a single vector, we isolated clones derived from transfections using the transposase and the transposon cassettes in cis (on the same plasmid vector) and found that all clones had residual transposase expression

In contrast, stable clones generated with transposase delivered in trans from a separate non-integrating plasmid showed no residual transposase expression Studies are ongoing to further evaluate the potential genotoxicity of piggyBac in human cells Thus, our data suggests that piggyBac transposase expression is short lived (7 days)

in a mixed population of human cells, the piggyBac transposase appears incapable of mobilizing TRS-like-sequences in the human genome, and delivering transposase in trans should be safer than delivery in cis when modifying human cells piggyBac appears to be

a promising non-viral gene delivery system for therapuetic genetic modifi cation of human cells

Transposon-Based Integration System for Gene Transfer in Human Epithelial Cells

Giandomenico Turchiano,1 Maria Carmela Latella,1 Zsuzsanna Izsvak,2 Zoltan Ivics,2 Fulvio Mavilio,1 Alessandra Recchia.1

1 Center for Regenerative Medicine, University of Modena and Reggio Emilia, Modena, Italy; 2 Max Delbruck Center for Molecular Medicine, Berlin, Germany.

Transplantation of autologous, genetically corrected epidermal stem cells (EpSC) was successfully used to treat junctional epidermolysis bullosa (EB), a genetic skin adhesion disorder The dystrophic forms of EB is caused by mutations in the type-VII collagen gene (COL7A1) Delivering the >9 kb COL7A1 cDNA by

a retroviral vector is problematic, due to the large size and highly repeated nature of its sequence, which induce genetic rearrangements during reverse transcription and integration We tested the feasibility

of using a non-viral vector system based on Sleeping Beauty (SB)-derived transposons, taking advantages of the recently developed, high-capacity “sandwich” version of the SB transposon and the

“hyperactive” SB 100X transposase, which showed high transposition effi ciency in human stem cells We tested the system in HeLa cells and in a keratinocyte cell line (HaCaT), which were co-transfected with the SB 100X transposase and either the normal or the sandwich version of the SB transposon containing a small-size reporter gene (Venus or GFP) expression cassette In both cell lines, transposition was obtained in up to 80% of the transfected cells with the sandwich transposon, compared to ∼50% obtained with the older version Transposed HaCaT cells were cloned and analysed for integration events Individual clones carried one to 14 copies of the integrated transposon of the predicted size High-throughput sequencing is under way to analyze the sandwich SB transposon integration characteristics

We then tested a transposon carrying an 8.8-kb cassette, which again showed up to 80% transposition effi ciency in transfected cells Finally,

we generated sandwich transposons containing an expression cassette for the COL7A1 cDNA under the control of a constitutive (PGK) or a keratinocyte-specifi c (K14) promoter, which are currently being tested for integration in HaCaT cells The SB-based gene delivery system will fi nally be tested in human primary keratinocyte cultures