96 The Use of a Mutant RNA Polymerase II Largest Subunit That Confers α Amanitin Resistance as a Selection Marker for Ex Vivo Gene Therapy Molecular Therapy �������� ��� ���� ���������������� �������[.]
Trang 1Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy
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94 Recombinant Sendai Virus-Mediated
CFTR cDNA Transfer
Stefano Ferrari,1,4 Raymond Farley,1,4 Felix Munkonge,1,4 Uta
Griesenbach,1,4 Stephen N Smith,1,4 Jun You,2 Tsuyoshi
Tokusumi,2 Akihiro Iida,2 Brandon Wainwright,3 Mike Gray,5
Angela Wright,5 Bernard Verdon,5 Barry Argent,5 Duncan M
Geddes,1,4 Mamoru Hasegawa,2 Eric W F W Alton.1,4
1 Gene Therapy, NHLI,Imperial College, London, United
Kingdom; 2 DNAVEC Research Inc, Tsukuba, Ibaraki, Japan;
3 Department for Molecular & Cellular Biology, University of
Queensland, Brisbane, Australia; 4 UK Cystic Fibrosis Gene
Therapy Consortium, United Kingdom; 5 School of Cell &
Molecular Biosciences, Newcastle University, Newcastle, United
Kingdom.
Recombinant Sendai virus (SeV) can deliver reporter and
therapeutic genes to the airway epithelium very efficiently To assess
applicability for Cystic Fibrosis (CF), we cloned the human CFTR
cDNA into the SeV genome between the Matrix and the Fusion
genes CFTR activity was assessed on C127-/- using a radioactive
iodide efflux assay and results expressed as change in the apparent
rate constant per minute (Δk min-1) ± s.e.m Positive controls (T84
cells) showed maximal efflux rate 2 min after forskolin/IBMX (F/I)
addition (0.24 ± 0.02, n=30) compared to C127-/- values (0.004 ±
0.004, n=11), p<0.001 SeV-CFTR showed a peak 3 min after F/I
addition (0.34 ± 0.04, n=12) As for untreated C127-/- cells,
treatment with SeV carrying a mutated CFTR (SeV-mCFTR) did
not lead to any change in efflux (-0.01 ± 0.02, n=12) Whole cell
perforated patch-clamp showed F/I-stimulated current in
C127-/-infected with SeV-CFTR (10/11), but with SeV-LacZ (0/7) The
SeV induced current had a linear current/voltage relationship and
as expected for CFTR In vivo experiments were carried out in
G551D CF transgenic mice by administration of SeV-CFTR (5 x
107 pfu, n=9) through nasal perfusion and CFTR activity analysed
at days 2,7,14,28 At a similar titre, 100% of nasal epithelial cells
expressed the reporter gene when LacZ was perfused
SeV-mCFTR (n=9) was used as control Two days after infection, NPDlow
Cl- values in animals treated with SeV-CFTR (-0.1 ± 1.0 mV) were
significantly (p<0.05) higher (i.e., towards non-CF values) than
those observed in animals treated with SeV-mCFTR (-3.3 ± 0.7
mV) SeV-CFTR treated values remained significantly (p<0.05) higher
at day 7 (0.2 ± 0.7 mV) compared to –1.6 ± 1.2 mV for controls
Baseline PD values at day 7 for animals treated with SeV-mCFTR
(30.8 ± 3.6 mV) were not different to untreated CF mice, but in mice
treated with SeV-CFTR they were not different from those observed
in wild-type mice (12.1 ± 1.5 mV, p<0.01 compared to controls)
By day 28, baseline and low chloride values had reverted to typical
G551D CF mouse values Since overexpression of CFTR can lead
to cell abnormalities in a further series of SeV vectors CFTR gene
was positioned towards the 3’-end (polar effect) in order to lower
the CFTR expression A reduced F/I-stimulated efflux was seen
when CFTR was positioned between the HN and L genes (with the
L gene start signal) and after the L gene (with the F gene start signal)
Surprisingly when CFTR was positioned after the L gene but with
the NP gene start signal, CFTR activity was found to be higher than
observed with SeV-CFTR thus suggesting an interaction of the start
signals with the polar effect Experiments are underway to evaluate
the ability of these SeVs to correct the chloride/sodium defect in CF
mice In conclusion we have shown that SeV can mediate CFTR
gene transfer both in vitro and in vivo, the latter suggesting change in
both the sodium and chloride abnormalities
S Ferrari, U Griesenbach, D Geddes, E Alton and the company
DNAVEC are inventors of a patent covering the use of recombinant
SeV as a new gene transfer agent
95 Early Detection of 2-LTR Junctions in Mo-MLV Infected Cells Cytoplasm
Fatima Serhan,1 Magalie Penaud,1 Caroline Petit,2 Ghislaine Duisit,1 Pierre Sonigo,2 Philippe Moullier.1
1 Inserm ERM 01-05, CHU-Hotel Dieu, Nantes, France; 2 Inserm U567, Institut Cochin, Paris, France.
Unlike HIV, the nuclear import of the MLV preintegration complexes (PIC) requires mitosis of the infected cell for successful nuclear translocation and subsequent integration of the viral linear DNA A fraction of the reverse transcribed nuclear material circularizes resulting in the formation of 1 or 2-LTR dead-end products uneligible for integration These circles are believed to be generated in the nucleus and therefore are generally accepted as appropriate surrogate for an effective nuclear PIC translocation
We previously reported a producer cell line-dependent restriction
of amphotropic MLV replication characterized by a defective nuclear import of the reverse transcribed viral DNA To further investigate this observation, we analyzed the Mo-MLV viral DNA early after cell entry in the cytoplasm Since nondividing cells cannot support the nuclear translocation of PIC and the formation of 2-LTR circles,
we used aphidicolin-arrested MLV-infected cells as a control Total DNA was extracted at different time points after viral entry post infection and submitted to PCR analysis with primers allowing either the amplification of the late reverse transcribed viral DNA or the U5/U3 junctions Cell arrest in G1/S was confirmed by flow cytometry analysis
As soon as 2 hr post infection with a DNAse-treated Mo-MLV-containing supernatant, an unpredicted U5/U3 junction was reproducibly detected in aphidicolin-arrested NIH3T3-infected cells
as well as in non-arrested cells Negative controls consisted of AZT-treated cells or using an envelope-deleted virus Importantly, several cloned PCR products were sequenced and all corresponded to conventional described U5/U3 junctions Additionally, these early 2-LTR junctions were detected with different PCR primers also scanning for the U5/U3 region and in two other human cell types, i.e TE671 and ARPE
To determine the cellular location of the 2-LTR junctions, we next separated the cytoplasm and nucleus compartments of Mo-MLV-infected TE671 cells at different time intervals as soon as 1 hr post virus entry Viral DNA from the cytoplasmic fraction was analyzed
by PCR and Southern after purity assessment using a sensitive β-globin PCR/Southern assay Our data indicated that early 2-LTR junctions are reproducibly found in the cytoplasm We are currently investigating whether this viral DNA product is linear or circular or both and whether it is a replicative intermediate
96 The Use of a Mutant RNA Polymerase II Largest Subunit That Confers α αα αα-Amanitin Resistance as a Selection Marker for Ex Vivo Gene Therapy
N P van Til,1 J Seppen,1 R P J Oude Elferink.1
1 Experimental Hepatology, AMC Liver Center, Amsterdam, Noord-Holland, Netherlands.
Lentiviral vectors can transduce non-dividing cells for ex vivo
gene therapy It is desirable that 100% transduced cells are transplanted Unfortunately, some cell types such as hepatocytes, are not transduced with 100% efficiency and selection is necessary The ideal selection marker would work in non-dividing cells and would be non immunogenic
To circumvent the problem of an immunological response once the cells are transplanted we chose to use a mutant human RNA
polymerase II largest subunit (mhRPII) as an ex vivo selection marker.
This mutant cannot bind α-amanitin and therefore confers resistance
to this potent cytotoxin
Trang 2Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts
The cDNA corresponding to the coding sequences of mhRPII
was inserted downstream of the pgk promoter in a lentivector
followed by a myc-tag This lentiviral vector was used to produce
VSV-G pseudotyped virus
H35 rat hepatoma cells were transduced with lentivirus expressing
GFP (control) and lentivirus expressing mhRPII After 4 days
medium was refreshed and the cells were incubated with different
concentrations of α-amanitin Subsequently, after 4 days cells were
fixed and stained and colonies were counted
Control GFP lentivirus transduced H35 rat hepatoma cells
incubated with 5μM α-amanitin were all killed after 4 days In H35
cells transduced with mhRPII lentivirus, resistant colonies were
selected at 20μM α-amanitin These cells could easily be grown in
myc-tagged mhRPII protein expression
Finally, dividing and quiescent human fibroblasts were transduced
α-amanitin selection is possible in non-dividing cells
Both dividing and quiescent human fibroblasts were resistant
following transduction with mhRPII lentivirus Non transduced cells
were all killed
We conclude that transduction with mhRPII lentivirus can render
both dividing and non-dividing cells resistant to α-amanitin Because
mhRPII is an endogenous protein it has the advantage that an
immunological response to transduced cells will not occur after
transplantation
97 Metababolic Correction of MPS IIIB
(Sanfilipo Syndrome) Using Third Generation
Lentiviral Vector
Beth E Larson,1 Meg S Joesting,1 Nikunj V Somia,2 Tal Kafri,3
Chester B Whitley.1
1 Gene Therapy Center, Department of Pediatrics and Institute of
Human Genetics, University of Minnesota, Minneapolis, MN;
2 Molecular Cell Biology, University of Minnesota, Minneapolis,
MN; 3 Gene Therapy Center, University of North Carolina at
Chapel Hill, Chapel Hill, NC.
Sanfilippo syndrome type B, or mucopolysaccharidosis (MPS)
IIIB, is a lysosomal storage disease caused by systemic deficiency
Humans affected with MPS IIIB have a partial response to bone
marrow transplantation (Am J Med Genet 62:53, 1998), but do not
have a satisfactory neurologic outcome (Bone Marrow Transplant
15:S176, 1995) Current studies (Pan et al., this volume) have
suggested that VSV-G pseudotyped lentiviral vectors may provide
a better treatment in at least one MPS disease, including prevention
of neurologic damage A third-generation (4 plasmid) lentivirus was
chosen because of the safety of the self-inactivating vector and was
designed to express the human NAGLU cDNA under the
transcriptional control of the human PGK promotor Vector
preparations were prepared from 293T cells (plated at a density of
standard calcium phosphate procedure Media was changed 24
hours after co-transfection and the supernatant was collected 48
hours later Vector was concentrated with 2 rounds of centrifugation
and resuspended to a total volume of 1/1,000 of the original volume
A titer assay was performed using real-time PCR for the minus
strong-stop cDNA of encapsulated lentiviral particles (Scherr et al.,
BioTechniques 31:520-526) Titers were found to be in the range of
1 X 106 – 109 vector particles/ml After co-transfection, 293T cell
lysates were found to have increased NAGLU enzyme activity
Ongoing studies will determine the response of knockout mice
affected with Sanfilippo syndrome
98 Multiple Factors Influence Lentiviral Transduction Efficiency of Stem Cells
Elisabeth H Javazon,1 Miguel Sena-Esteves,1 Philip W Zoltick,1
Kirstin J Beggs,1 Jessica C Tebbets,1 Alan W Flake.1
1 General Surgery, Children’s Hospital of Philadelphia, Philadelphia, PA.
Adult derived mesenchymal stem cells (MSCs) demonstrate great potential as vehicles for gene therapy MSCs are easily isolated and
expanded in vitro and have been observed to engraft and differentiate
in vivo The development of gene transfer technology for MSCs
will not only advance current understanding of MSC engraftment, but will also significantly enhance the prospect of MSC based gene therapy Transduction of adult murine mesenchymal stem cells (AmMSCs) using different pseudotypes of lentiviral vectors has not been examined previously Here we compared the transduction efficiency of various pseudotypes of lentiviral constructs at a range
of multiplicity of infection (MOI) in AmMSCs All lentiviral pseudotypes consisted of the CS-CGW vector, which contained the WPRE and CPPT with GFP driven by an internal CMV promoter MSIN-VSV-G is similar to the CS-CGW vector except
for an extensive deletion in the right U3 region leaving only the att
sequence Our results demonstrated significant variability in transduction efficiency between lentiviral pseudotypes At the highest MOI, the rabies pseudotype transduced less than 10% of the AmMSCs, whereas the MSIN-VSV-G pseudotype transduced more than 95% of the AmMSCs Interestingly, MSIN-VSV-G pseudotype transduced significantly more AmMSCs than the
VSV-G pseudotype VSV-GFP expression remained stable and persisted for more than 12 passages in MSIN-VSV-G transduced AmMSCs Effective transduction of AmMSCs using lentiviral vectors was dependent upon MOI, lentiviral pesudotype, and an intrinsic component of the viral genome Comparisons of surface epitope expression, proliferation, and differentiation between transduced and nontransduced AmMSCs will be assessed to determine if
transduction alters AmMSC biology in vitro.