23 Cleavage/Excision of Plasmid DNA In Vivo Leads to Increased Maintenance and Persistence of Transgenes Expression in Mouse Molecular Therapy �������� ��� ���� ���������������� �������� ���� ������©[.]
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NAKED DNA: METHODS
21 Molecular Evidence for Sleeping
Beauty-Mediated Transposition and Long-Term
Expression In Vivo
Paul R Score,1 Joel L Frandsen,1 Jennifer Jeske,1 Perry B
Hackett,1,2 David A Largaespada,1 R Scott McIvor.1
1 Gene Therapy Program, Institue of Human Genetics, Dept of
Genetics, Cell Biology and Development, University of Minnesota,
Minneapolis, MN; 2 Discovery Genomics Inc., Minneapolis, MN.
Long-term expression is most effectively achieved through stable
integration of expression constructs into genomic DNA, preventing
degradation and ensuring transfer to future progeny The Sleeping
Beauty Transposon system (SB) offers a non-viral means to achieve
long-term gene expression by mediating efficient integration of
gene-carrying transposons Using a hydrodynamic injection technique to
deliver DNA to the livers of mice, several groups have shown that
the Sleeping Beauty system can be used to achieve long-term in vivo
expression However, in the experiments it is often difficult to
distinguish expression from episomal, integrated, or transposed
elements To better understand SB’s efficiency at mediating in vivo
transposition, we have designed a system to silence expression from
non-transposed elements and thus reduce or remove expression from
episomal or randomly integrated plasmids A transposon plasmid
pT2F/Cage (a transposon carrying a murine erythropoietin (epo)
gene transcriptionally regulated by a CMV enhancer / b-actin
promoter combination) was engineered to contain loxP sites
positioned to interrupt expression upon Cre-mediated recombination
Upon transposition these sites become segregated, thus protecting
the expression construct from Cre-mediated recombination and
subsequent silencing Mx1Cre inducible mice were injected with
pT2F/Cage with or without transposase-encoding plasmid At 2 to
4 weeks post-injection, in the absence of transposase, Cre induction
reduced the expression of epo to about 1% of that seen in the group
that included the transposase plasmid, which maintained epo levels
near that of the uninduced groups These results indicate a substantial
level of DNA-mediated expression not associated with transposition,
but which can be quantitatively distinguished from transposition
by its sensitivity to Cre recombinase They also provide additional
evidence for the effectiveness of the Sleeping Beauty transposon
system as an in vivo DNA-mediated gene transfer strategy for
achieving long-term expression We anticipate that this adaptation
of the LoxP-Cre recombinase system will be extremely useful for
the study of in vivo transposition as a gene therapy strategy, whether
mediated by Sleeping Beauty or by other transposon systems.
P.B Hackett, D.A Largaespada, and R.S McIvor have a financial
interest in Discovery Genomics Inc
22 Development and Analyses of Hyperactive
Forms of the Sleeping Beauty Transposase
Stephen R Yant,1 Jacob G Mikkelsen,1 Jason Hoyt,1 Hui Xu,1
1 Pediatrics and Genetics, Stanford University School of Medicine,
Stanford, CA, United States.
Previously, we have shown that nonviral and adenovirus-based
vectors encoding the Sleeping Beauty (SB) transposase/transposon
system support the stable integration of therapeutic genes in as
many as 6% of mouse hepatocytes in vivo Genomic integration
occurs via a highly ordered process called DNA transposition and is
triggered by the physical association of transposase monomers that
are bound to transposon end sequences In this report, we have
attempted to improve the overall efficiency of SB-mediated
transposition in mammalian cells by altering the affinity of
transposase subunits to itself and/or to the protein binding sites
located within the transposon terminal repeats Through site-directed mutagenesis, we have produced a total of ninety-six different transposase mutants, each containing a single amino acid substitution within the N-terminal DNA binding and subunit multimerization
domains of the SB transposase gene These mutants were cloned
into plasmids to permit high-level mammalian expression and then tested for their ability to support transposition of a neomycin-marked transposon following co-transfection and G418-resistant growth selection in HeLa cells Results indicate that twenty-one of these transposase mutants were ~1.5-to-4-fold more active than wild-type transposase, whereas nineteen other mutants were completely defective for transposition These results suggest that
many of these residues play an important role in Sleeping Beauty’s
biological activity Currently, we are using a yeast-based reporter system to investigate the relative DNA binding affinities of these mutants in an attempt to achieve a better understanding of the molecular mechanism(s) of hyperactivity In order to test whether the combination of individual hyperactive mutations could be used
to further improve the level of transposition in host cells, we have analyzed a small subclass of transposase ‘double’ mutants using our HeLa cell transposition assay Interestingly, some combinations
of these hyperactive mutations had an additive effect on transposase activity while other combinations produced transposase proteins that were less active relative to each of the single mutants These
results suggest that efficient SB-mediated transposition may require
a delicate balance between assembly of a stable transposase/DNA complex and rapid dissociation of this complex during re-insertion
of excised elements Although additional changes to the transposase
gene are ongoing, we have already obtained two SB mutants which
are 5-to-8 times more active than the wild-type transposase In addition, we have recently generated a modified transposon vector that exhibits a two-fold improvement in transposition in HeLa cells compared to the wild-type vector We are presently combining all of these improvements and are studying the activity of this new
hyperactive system in vivo following hydrodynamic-based delivery
to mouse liver In summary, our research demonstrates the potential for transposase evolution and suggests that dramatic improvements
in the integration frequencies of transposon-based vectors will be readily obtainable These improvements should greatly extend the utility of this integrating vector system for both basic research and human gene therapy applications
23 Cleavage/Excision of Plasmid DNA In Vivo Leads to Increased Maintenance and Persistence
of Transgenes Expression in Mouse
Efren Riu,1 Zan Huang,1 Mark A Kay.1
1 Pediatrics and Genetics, Stanford University School of Medicine, Stanford, CA.
Persistence of transgene expression is a major limitation for non viral mediated gene therapy approaches We have recently established that bacterial DNA sequences can variably affect transcription of
transgenes in vivo To establish if covalent linkage of the bacterial
DNA to the transgene was critical for transcriptional repression, we analyzed whether cleavage/excision of bacterial plasmid DNA
sequences in vivo influenced the long-term transgene expression in
mouse liver To do this, the human alpha-1-antitrypsin (hAAT) and human clotting factor IX (hFIX) reporter genes were flanked by
two I-SCeI meganuclease recognition sites, and co-injected together with a plasmid encoding the I-SCeI cDNA or a control plasmid
DNA into mouse liver Two weeks after DNA administration, mice injected with the reporter gene alone or with the irrelevant control plasmid showed very low serum levels of hAAT and hFIX, which remained low throughout the length of the experiment However,
animals that expressed I-SCeI had a 5-10 fold increase in serum
hAAT and hFIX that persisted for at least 8 months (length of
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Copyright © The American Society of Gene Therapy
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study) In addition, expression of I-SCeI resulted in cleavage and
excision of hAAT and hFIX expression cassettes from plasmid
backbone, as established by Southern blot analyses This suggested
that covalent linkage of bacterial DNA markedly diminished
long-term transgene expression in vivo Therefore, to increase the
persistence of transgenes in vivo and, thus, optimize non viral gene
therapy approaches, the excision of the transgenes from plasmid
backbone and the removal of bacterial DNA should be considered
24 New Insight into Cellular Uptake of
Nucleic Acids: A Role for the Nucleic
Acid-Conducting Channel
Edgar Leal-Pinto,1 Avelino Teixeira,1 Scott C Henderson,2 Mary
E Hawkins,3 Paul E Klotman,1 Basil Hanss.1
1 Nephrology/Medicine, Mt Sinai School of Medicine, New York,
NY, United States; 2 Molecular, Cell, and Developmental Biology,
Mt Sinai School of Medicine, New York, NY, United States;
3 Pediatric Branch, National Cancer Institute, NIH, Bethesda, MD,
United States.
Much of the published literature suggests that receptor-mediated
endocytosis is the primary mechanism of cellular uptake of
oligodeoxynucleic acids (ODN) In many of these studies ODNs are
labeled with a large, hydrophobic fluorophore such as FAM (a
derivative of fluorescein) We have hypothesized that the presence
of a large hydrophobic molecule on the ODN would change the
kinetics, if not the mechanism, of uptake To test this hypothesis
we developed the methodology to examine ODN uptake in cultured
cells using a new structural analog of 2-deoxyguanosine,
3-Methyl-8-(2-deoxy-β-D-ribofuranosyl) isoxanthopterin (3MI) 3MI is highly
fluorescent and is very closely related in structure to guanosine As
such, it does not introduce significant structural or steric changes to
an ODN For these studies, ODN was labeled with 3MI or FAM
(FAM-ODN) and comparisons of uptake kinetics in LLC-PK1 cells
(a renal epithelial cell line derived from pig kidney) were made using
multi-photon laser scanning microscopy The rate of 3MI-ODN
uptake is significantly (p<0.05) higher than that of FAM-ODN,
suggesting that FAM labeled- and 3MI labeled-ODNs are transported
by different mechanisms The kinetics of FAM-ODN uptake
suggested a single carrier model with a time constant consistent with
endocytosis 3MI-ODN uptake was best fit by a multicarrier model
with two components, a fast component and a slow component
Based on these data, we hypothesized that FAM labeled ODNs
gain entry to the cell exclusively via an endocytic pathway whereas
3MI-ODNs enter the cell via an endocytic pathway and by a fast
component that we hypothesize represents the nucleic acid channel
To test these hypotheses, uptake experiments were performed in
- 30 minute pre-incubation), a blocker of both clathrin- and
caveolae-mediated endocytosis FAM-ODN uptake was significantly
suggest that FAM-ODN is transported into the cell primarily by an
component of 3MI-ODN uptake whereas the slow component of
uptake was completely inhibited These data indicate that the slow
component of 3MI-ODN uptake is mediated by endocytosis To
determine if the fast component of 3MI-ODN uptake is mediated
by the nucleic acid channel (NACh), experiments were conducted
using the antiserum GN-2640, which blocks NACh in lipid bilayer
experiments When cells were pre-treated with GN-2640 (diluted
1:50) beginning 10 minutes prior to adding 3MI-ODN, the fast
component of ODN uptake was blocked, suggesting it is mediated
by NACh The slow endocytic component, however, remained intact
as indicated by a gradual increase in intracellular fluorescence
Pre-immune serum was without effect In summary, these data indicate
that FAM-ODN enters the cell primarily by endocytosis, whereas
3MI-ODN is internalized by the combined action of NACh and an endocytic pathway These data have led us to conclude that the nucleic acid channel is significant mechanism of ODN uptake in LLC-PK1 cells
25 Enhancement of Ultrasound-Mediated Transfection by the Combination of Cavitation Facilitation and Plasma Membrane Modification
In Vitro and In Vivo
Ryohei Ogawa,1 Tetsuo Nozaki,2 Go Kagiya,3 Loreto B Feril, Jr.,1 Hideki Fuse,2 Takashi Kondo.1
1 Department of Radiological Sciences, Faculty of Medicine, Toyama Medical and Pharmaceutical University, Toyama, Toyama, Japan; 2 Department of Urology, Toyama Medical and Pharmaceutical University, Toyama, Toyama, Japan; 3 Medical Division, The Wakasa Wan Energy Research Center, Tsuruga, Fukui, Japan.
We previously reported that an echo contrast agent, Levovist, enhanced the effect of ultrasound-mediated transfection (USMT) in vitro (Ultrason Sonochem, 9: 197 (2002)) The mechanism underlying USMT is possibly explained by the interaction between the effects of inertial cavitation associated with ultrasound and a response of the plasma membrane to its effects, although the real mechanism has not been completely elucidated In this study we focused on the plasma membrane to see if altering it could enhance USMT in vitro and in vivo
PC-3 cells from a prostate cancer and T24 cells from a bladder cancer were used as target cells A plasmid with the luciferase gene under control of a CMV promoter was used as a reporter To modify plasma membrane, lidocaine, a local anesthetic, or heat was applied Levovist was used to facilitate cavitation generation In vitro, cells in a rotating polystylene tube were sonicated in a water bath at various temperatures with 1 MHz continuous ultrasound at
3 W/cm² for 20 sec In vivo, cells were transabdominally sonicated after cell suspension was transurethrally introduced in the bladder
of a 4-week old female Wister rat with 1 MHz ultrasound at 2 W/ cm² with 30% duty cycle for 60 sec Sonicated cells were incubated
at 37°C for 24 hrs before lysing and subjecting to lucifearase assay
In the case of in vitro transfection of PC-3 cells, luciferase expression increased with increasing lidocaine concentration and also increased with rising temperature only when Levovist was present In the presence of 10 mg/ml of Levovist, we observed 17-fold higher efficiency in USMT with 1 mM lidocaine and 18-17-fold at 44°C in comparison with that observed without lidocaine at 37°C The increase in reporter gene expression was not due to induced promoter activity since the increase was not observed when lidocaine
or heat was applied immediately after sonication Both treatments were shown to increase plasma membrane fluidity though at different degrees In addition, heat significantly facilitated cavitational effect determined by free radical detection on ESR, but lidocaine did not, suggesting different mechanisms of USMT enhancement by lidocaine and heat
In the case of in vivo transfection of PC-3 cells, we observed similar results to those of the in vitro study Detected luciferase activities increased dose-dependently with Levovist (50 to 200 mg/ ml) When 1 mM lidocaine was added to the cell suspension in addition to 100 mg/ml Levovist, detected luciferase activity increased about 5-fold of that without lidocaine Likewise, when a rat was incubated in a water bath at 42°C prior to sonication with the bladder instilled with cell suspension containing 100 mg/ml Levovist, the luciferase activity was raised to 9-fold of that sonicated without preincubation T24 cells showed similar results though to a lesser degree