Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 466-477
doi:10.1111/j.1745-7270.2008.00430.x
Experimental cancer gene therapy by multiple anti-survivin
hammerhead ribozymes
Qi Fei1,2, Hongyu Zhang2, Lili Fu2, Xinlan Dai2, Baomei Gao2, Min Ni2, Chao Ge2, Jinjun Li2, Xia Ding3, Yuwen Ke3, Xuebiao Yao3*, and Jingde Zhu2*
1 Fudan University School of Medicine,
Shanghai 200032, China
2 Cancer Epigenetics and Gene Therapy Program,
The State Key Laboratory for Oncogenes and Related Genes, Shanghai Cancer
Institute, Shanghai Jiaotong University, Shanghai 200032, China
3 Division of Cellular Dynamics, Hefei
National Laboratory, University of Science and Technology of China, Hefei
230027, China
Received: April 6,
2008
Accepted: May 5,
2008
This work was
supported by the grants from the Shanghai Science Foundation (No. 07DJ14074 to
JZ), the National Science Foundation (Nos. 30570850 and 10574134 to JZ), the National
Natural Science Foundation of China (No. 2004CB518804 to JZ and No.
2002CB713700 to XY), the National Basic Research Program of China (Nos.
2006AA02Z320 and 2006AA02Z197 to JZ), and the European 6th Program (No. LSHB-CT-2005-019067
to JZ). XY is a GCC Eminent Scholar
*Corresponding
authors:
Jingde Zhu:
Tel/Fax, 86-21-64224285; E-mail, [email protected]
Xuebiao Yao:
Tel/Fax, 86-551-3607141; E-mail, [email protected]
To improve the efficacy of gene therapy for
cancer, we designed four hammerhead ribozyme adenoviruses (R1 to R4) targeting
the exposed regions of survivin mRNA. In addition to the in vitro
characterization, which included a determination of the sequence specificity of
cleavage by primer extension, assays for cell proliferation and for in vivo
tumor growth were used to score for ribozyme efficiency. The resulting
suppression of survivin expression induced mitotic catastrophe and cell death
via the caspase-3-dependent pathway. Importantly, administration of the
ribozyme adenoviruses inhibited tumor growth in a hepatocellular carcinoma
xenograft mouse model. Co-expression of R1, R3 and R4 ribozymes synergistically
suppressed survivin and, as this combination targets all major forms of the survivin
transcripts, produced the most potent anti-cancer effects. The adenoviruses
carrying the multiple hammerhead ribozymes described in this report offered a
robust gene therapy strategy against cancer.
Keywords hammerhead ribozyme; survivin;
hepatocellular carcinoma; gene therapy
Defects in apoptosis are a key hallmark of the malignant cells
resulting from such factors as the activation of anti-apoptotic genes and the
repression of pro-apoptotic genes [1]. A well-known anti-apoptotic factor is
survivin [2], which is overexpressed in all types of cancers but is
undetectable in normal adult cells. Survivin participates in a variety of
cellular processes such as cell division, angiogenesis, apoptosis etc [3].
Survivin negatively regulates apoptosis by interfering with
caspase-9-processing [4]. Despite our early findings that it binds to
Smac/DIABLO released from mitochondria [5], how survivin directs crossroad
traffic between cell division and apoptosis remains elusive.Besides serving as a biomarker for cancer diagnosis and prognosis
[6,7], survivin has been selected as a target for cancer intervention [3].
Forced expression of CDK1-nonphosphorylatable survivinT34A
mutant has displayed dominant-negative effects and induces dramatic cell death
[8,9]. Down-regulation of survivin expression by antisense oligonucleotide has
exhibited a promising outcome in xenograft models and phase I clinical trials
[10,11]. Other down-regulation approaches involving RNA interference (RNAi) also
have produced therapeutic effects in xenograft models [12–14]. However,
there has been a long debate as to the specificity and durability of antisense
oligonucleotide and RNAi approaches in clinical therapy.Ribozyme is a powerful new therapeutic tool used to diminish RNA
molecules in cells [15]. The hammerhead ribozyme is the smallest and best
characterized. It consists of three parts: an invariant catalytic domain with
13 conserved nucleotides in a stem-loop structure and two arms located on each
side of the catalytic domain stretched complementary to the target RNA.
Hammerhead ribozymes cleave after NUH (where N can be any nucleotide, and H can
be any nucleotide except G) sequences of target mRNA to suppress the target
protein expression [16]. The substrate specificity of the
ribozyme is determined by the paired regions flanking the cleavage site.
Binding of ribozymes to its target mRNA is significantly more stringent than
short interfering RNA approaches, which often produce off-target effects [17,18].
We have used the expression cassette in pGVaL to ensure a high level
of expression of the stable ribozyme RNAs for better physical contact with
substrate RNA [19]. The substrate RNA has been systematically assessed in
vitro, in cell cultures and in mouse tumor xenograft model systems with the
human hepatocellular carcinoma as the targeted tumor type.
Materials and Methods
Anti-survivin ribozyme expression plasmid vectorsThe coding region (the A of ATG: 1 to A of TGA: 426) of the human survivin mRNA sequence (GenBank accession No.
BC008718) was analyzed for exposed regions (loops) using the MFOLD program (http://mfold.bioinfo.rpi.edu/cgi-bin/rna-form1.cgi).
The ribozyme sequences targeting H of the NUH triplet in each of four loops
were designated R1 to R4 and +61, +83, +232 and +358, respectively (Fig. 1).
Each pair of the oligonucleotides (Table 1) were phosphorylated,
annealed and cloned at SalI and PstI sites of pGVaL plasmid
vector [19] [Fig. 2(A)]. The constructs were designated pGVaL-R1 to R4.
Survivin expression plasmid vectorThe survivin coding region (the A of ATG: 1 to A of TGA: 426)
was amplified by plaque-forming unit-polymerase chain reaction from the cDNA
from SMMC-7721 liver cancer cells with the primers SurL 5?-CCGctcgagatgggtgccccgacg-3? and SurR 5?-GAagatctatccatggcagccagct-3? (BglII and XhoI recognition sequences are underlined,
respectively). This was followed by cloning in the downstream phase of the
three hemagglutinin (HA) tag sequences of pcDNA-HA vector to form HA-survivin
(HAS). The HindIII (blunt) and XbaI luciferase fragment of
pGL-3-basic (Promega, Madison, USA) was cloned at the BglII (blunt) and XbaI
sites of HAS to generate the plasmid HA-survivin-luciferase (HASL) [Fig.
2(C)].
Non-replicative adenoviral-ribozyme vectors The BamHI/NheI (blunt) fragment of pGVaL and pGVaL-R1
to R4, was placed at BglII/HindIII (blunt) sites of pDC vector
using the AdMax system (Microbix, Calgary, Canada) to create pDC-GVaL and
pDC-R1 to R4, respectively [Fig. 2(B)]. The EcoRI/SacI
(blunt) fragment of pDC-R1 was placed at EcoRI/SmaI sites of
pDC-R3 to create pDC-R13. The EcoRI/SacI (blunt) fragment of
pDC-R13 was placed at EcoRI/SmaI sites of pDC-R4 to create
pDC-R134. The adenoviral particles were made by the CreLoxP (3CreLoxP) mediated
recombination between each pDC vector and adenoviral vector (pBHGE3?E1) in
HEK 293 cells. The sequenced adenoviro/ribozyme particles were amplified,
titered and kept at –20 ?C until used. The adenoviruses were batch-produced and purified
by Shanghai Sino-Gene Company (Shanghai, China).The BamHI/NheI (blunt) fragment of pGVaL and pGVaL-R1
to R4, was placed at BglII/HindIII (blunt) sites of pDC vector
using the AdMax system (Microbix, Calgary, Canada) to create pDC-GVaL and
pDC-R1 to R4, respectively [Fig. 2(B)]. The EcoRI/SacI
(blunt) fragment of pDC-R1 was placed at EcoRI/SmaI sites of
pDC-R3 to create pDC-R13. The EcoRI/SacI (blunt) fragment of
pDC-R13 was placed at EcoRI/SmaI sites of pDC-R4 to create
pDC-R134. The adenoviral particles were made by the CreLoxP (3CreLoxP) mediated
recombination between each pDC vector and adenoviral vector (pBHGE3?E1) in
HEK 293 cells. The sequenced adenoviro/ribozyme particles were amplified,
titered and kept at –20 ?C until used. The adenoviruses were batch-produced and purified
by Shanghai Sino-Gene Company (Shanghai, China).
In vitro analysis of the anti-survivin
ribozymesBoth ribozymes and HASL RNAs were made by the in vitro
transcription of 2 mg linearized pGVaL/pGVaL-R4 (by XbaI) and 2 mg HASL (by HindIII),
respectively, in vitro transcribed with T7 RNA polymerase (TaKaRa,
Dalian, China). This was followed by the removal of the DNA template by DNase
I. The transcripts were then purified and quantified by semi-quantitative
reverse transcription-polymerase chain reaction (RT-PCR) using a known amount
of plasmid DNA as a reference.For the in vitro ribozyme cleavage reaction of HASL RNAs,
equal molar amounts (1 mmol) of ribozyme and the HASL RNAs were heat denatured
in 10 ml of 50 mM Tris (pH 7.5)/1 mM EDTA, quickly cooled and followed by
the addition of MgCl2 for a final concentration of 10 mM to
initiate the cleavage reaction at 37 ?C for 60 min. The cleavage products were
NH4Ac/ethanol precipitated. They were analyzed for cleavage specificity
by primer analysis and for cleavage efficacy by determining the relative
luciferase activity and protein amount (Western blot analysis with the HA
antibody) of the in vitro translated products. The non-virus control
(mock) was made from the cleavage reaction with the in vitro transcribed
RNAs from pGVaL.Two-thirds of both mock and cleaved transcripts were subjected to
primer extension analysis with the 5?-end [32P]-labeled
DNA primer by T4 polynucleotide kinase [SPE1 for R1 and R2, SPE2 for R3 and
SPE3 for R4, Fig. 1(C)]. The sequence ladders were made with the same
primer, and the HASL template was created by T7 DNA polymerase-based
manual-sequencing (Amersham, Buckinghamshire, UK). The remaining one-third of the in
vitro cleavage products were in vitro translated (Rabbit
Reticulocyte Lysate System; Promega), one-fifth of which were measured for
luciferase activity (Promega) in a LB 9506 Lumat luminometer (EG&G,
Gaithersburg, USA) and plotted. The cleavage reaction by all four ribozymes was
also included in this analysis. The remaining four-fifths of the products were
analyzed with anti-HA antibody (Santa Cruz Biotechnology, Santa Cruz, USA) and
visualized by SuperSignal West Pico Chemiluminescent Substrate (Pierce,
Rockford, USA). The relative densitometric reading of the cleaved over that of
the mock (as 100%) was calculated.
Evaluation of the anti-survivin adeno/ribozyme effect in
hepatocellular cellsSMMC-7721 liver cancer cell line (Cell Bank No. TCHu68; China), and PLC
hepatocarcinoma cells (ATCC No. CRL-8024) were cultured at 37 ?C with 5% CO2 in Dulbeccos modified eagles medium (DMEM; Invitrogen, Carlsbad,
USA) supplemented with 10% fetal bovine serum (PAA, Pasching, Austria) or 10%
newborn bovine serum (Sijiqing, Hangzhou, China). The log-phase cells were
continuously infected for 72 h by each virus followed by a
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reading for
cell viability. The relative MTT reading was calculated by the following formula.
The cells were collected at 48 h for RT-PCR and Western blot analysis.
Eq.
The semi-quantitative RT-PCR analyses were carried out for survivin,
b-actin and ribozymes, respectively, with the primer pairs (Table
2). The expression level of the survivin mRNA was presented as a
ratio (the survivin over b-actin densitometric quantification) of the infected cells over the
mock control. Western blot analyses of survivin (anti-survivin antibody; Santa
Cruz Biotechnology) and proliferating cell nuclear antigen (PCNA) (anti-PCNA
antibody; Santa Cruz Biotechnology) were performed on a 15% SDS-polyacrylamide
gel. Western blot analysis of poly(ADP-ribose) polymerase (PARP) (85 kDa;
Promega)/PCNA was carried out on a 7.5% SDS-polyacrylamide gel. The relative
abundance of the survivin or PARP protein was presented in the density ratio of
that over PCNA of the viral-infected cells over that (arbitrarily as 100%) of
the non-infected cells.
Immunofluorescence microscopySMMC-7721 cells were infected with the control (GVaL) and survivin
ribozymes (R3 or R134), respectively, followed by fixation in 4% formaldehyde
24 h after infection. Coverslips were then permeabilized with 0.2% Triton X-100
and blocked with PBS containing 0.05% Tween-20 with 1% bovine serum albumin
(Sigma, St. Louis, USA). Cells were incubated with primary antibodies (tubulin
mouse antibody DM1A, 1:5000 dilution; human anti-centromere antibody ACA,
1:2000 dilution) in a humidified chamber for 1 h and then washed three times in
PBS containing 0.05% Tween-20. Texas red-conjugated goat antihuman IgG+IgM and
fluorescein-isothiocyanate-conjugated rabbit antimouse IgG (Jackson ImmunoResearch,
West Grove, USA) were used as the secondary antibodies for visualization of
appropriate antigens. DNA was stained with TOTO-3 dye (Invitrogen). Slides were
examined with a Leica SP5 confocal microscope (Leica, Heerbrugg, Switzerland)
and images were presented with Photoshop.
Inhibitory effect of the anti-survivin adeno/ribozyme on tumor
xenograft modelThe log-phase SMMC-7721 cells were infected with the virus at
multiplicity of infection (MOI) of 10 or 25 for 24 h at 37 ?C before being
injected subcutaneously into both flanks (3?106 cells/flank) of each mouse (BALB/c, nu/nu).
Each group consists of three mice; the left flank of each mouse was injected
with SMMC-7721 cells or adeno/GVaL (non-ribozyme viral control) while the right
flank was injected with the cells infected with each adeno/ribozyme virus under
study. Tumor growth was monitored and measured until the experiment terminated
at week 4, when the tumor mass was measured. The weight ratio of the tumor over
the mock was determined for each mouse. All animal experiments were approved by
the Institutional Review Board and the Animal Research Committee.The tumor was fixed, sectioned, hematoxylin and eosin (H&E)
stained or immunostained for the expression of survivin (1:10 dilution of
anti-survivin antibody; FL-142, Santa Cruz Biotechnology) and ki67 protein
(1:10 dilution of anti-Ki67 antibody; Novocastra, Newcastle, UK), and
visualized by the EnVision System (Dako, Carpinteria, USA).
Results
Design of the anti-survivin hammerhead ribozymes Four hammerhead ribozymes were designed to cleave respectively at
nucleotide +61 (R1), +83 (R2), +232 (R3), and +358 (R4) of the predicted open
frames of survivin mRNA (Fig. 1). The genes were then embedded in
the adenoviral Va I RNA of the pGVaL vector [19] [Fig. 2(A)]. By placing
the ribozyme genes at the center of a loop formed from an inverted repeat
sequence that was artificially introduced into the Val I gene [19]
[pGVaL-Rx in Fig. 2(A)], the ribozyme sequences were expected to
protrude from the Va I RNA region. The BamHI and NheI fragment of
each pGVaL-R was cloned into the adenoviral vector (Microbix pDC-316 based) to
facilitate analysis of both the cellular level and the entire animal and that
will ultimately be used to treat cancer in clinical trials [Fig. 2(B)].
The HA-tagged survivin-luciferase fusion gene was put into a pcDNA 3.1-based
vector to create HASL [Fig. 2(C)], so that evaluation of the ribozyme
mediated cleavage in vitro of survivin RNA could be readily
carried out by primer extension (for both cleaving specificity and efficacy)
and luciferase activity (for cleaving efficacy).
Evaluation of survivin mRNAs ribozyme-cleavage specificity
and efficacy Except for R3, which failed to target DEx3, all the remaining
ribozymes were expected to cleave all the three major forms of survivin
mRNAs [20,21] [Fig. 3(A)]. Equal amounts of both ribozymes and the survivin
RNAs made by in vitro transcription were incubated in the presence of Mg2+ for the cleavage reaction. Primer extension analysis of the
resulted survivin RNAs showed that each ribozyme cleaved the survivin
RNA at the predicted sites [arrowed in Fig. 3(B)]. Judged from its band
density relative to its expected size, R2 appeared to be least efficient
ribozyme. The in vitro translated products of the RNA remaining from the
cleavage reaction were, respectively, assayed for luciferase activity [Fig.
3(C)] and for the size of the HA-associated protein band in Western blot
analysis [Fig. 3(D)] to semi-quantitatively determine the efficacy of
the ribozyme cleavage. The luciferase activity from the mock (100%) was reduced
to 62.7% in R1, 70.0% in R2, 40.4% in R3, 57.0% in R4, and 28.7% in a
combination of the four ribozyme experiments [Fig. 3(C)] and the
HA-associated band density to 48.8% in R1, 124.4% in R2, 28.7% in R3, 61.0% in
R4 and 16.5% in a combination of the four ribozyme experiments [Fig. 3(D)].
As the in vitro analyses indicated, R3 was the most effective, followed
by R1, R4 and R2. There was an additive effect in cleavage by all the four
ribozymes in combination [Fig. 3(C,D)]. In view of the protein-free RNA
in vitro analyses [Fig. 3], the exposed region of the substrate
RNA by the MFOLD program was indeed informative for the rational design of the
hammerhead ribozyme.
Ribozyme-mediated cleavage of survivin mRNA-induced apoptosis
and mitotic catastrophe in cells SMMC-7721 hepatocellular carcinoma cells were challenged with the
non-replicative adenoviral particles carrying a single ribozyme gene (R1 to
R4), three ribozymes (R1, R3 and R4) in tandem (R134) or no ribozyme control
(GVaL) for 72 h. The loss of cell viability then was assayed by MTT assay [Fig.
4(A)], by a semi-quantitative PCR analysis for repression of survivin
expression [Fig. 4(B)] and by Western blot analysis [Fig. 4(C)].
Western blot analysis was also used to assess the apoptotic index for the level
of PARP protein [Fig. 4(D)]. Consistent with the in vitro observations (Fig. 3), R2
was the least effective ribozyme; infection at MOI 50 reduced the MMT reading
by no more than 20%. R134 was more effective than the most potent single
ribozyme, R3; infection at MOI 12.5 reduced the MTT reading by 23.2% versus
38.6% and at MOI 25 by 15.2% versus 27.8% [Fig. 4(A)]. The levels of
ribozyme and b-actin RNAs were comparable, but the survivin mRNA level
varied with the ribozyme viruses: GVaL, 91.4% of that of the SMMC-7721 cells;
R1, 36.1%; R2, 108.3%; R3, 33.8%; R4, 60.5%; and R134, 22.0% [Fig. 4(B)].
These results reflected the relative potency of the ribozyme against survivin
mRNA, which was also confirmed by Western blot analysis of survivin protein [Fig.
4(C)]. PARP protein (85 kDa) was a specific cleaved product of PARP-1 by
caspase-3 in the cell undergoing apoptosis [22]; the proteins level increased
from 100% in mock infected cells to 164.0% by R2, 479.9% by R1, 768.8% by R3,
460.1% by R4 and 1339.3% by R134 in viral-infected cells [Fig. 4(D)]. We
also performed the same set of analyses on another hepatocellular cell line PLC
(ATCC No. CRL-8024) and yielded comparable results (Fig. 5).An immunofluorescence study showed that when survivin was depleted
of R3 and R134, respectively, SMMC-7721 cells exhibited aberrant chromosome
segregation [Fig. 4(E), d, d], a phenotype associated with
mitotic catastrophe. Multipolar spindles were typical phenotypes seen in 83%±7%
of the infected cells where mitotic chromosomes failed to align at the equator
and enter into anaphase [Fig. 4(E), c, c], compared to 5%±3% in
GVaL-infected control (n=4). The microtubule integrity and centromere
labeling by anti-centromere antibody ACA remained unaltered, confirming the
specificity of the ribozyme treatment [Fig. 4(E), a, a].
Adenoviral-ribozymes prevented and suppressed growth of the
hepatocellular tumor xenografts in mice SMMC-7721 cells were infected with viruses at MOI 25 for 24 h prior
to injection into mice. The right flanks of nude mice were injected with
SMMC-7721 cells that had been pre-infected with the ribozyme viruses, while the
left flank received cells with either no virus (mock) or the empty vector-virus
(GVaL) pre-infection [Fig. 6(A)]. While no difference in growth profile
and tumor mass was observed in the mock-infected SMMC-7721 cells [Fig. 6(B,D)],
all the ribozyme viruses, except for R2, prevented tumor growth. The tumors
from the cells pretreated with R2, the least effective ribozyme, grew
significantly more slowly than those pretreated with the mock, and at 35 d when
the experiment was ended, had a smaller mass by a ratio of 0.64: 1 [Fig.
6(C,D)]. Again neither R3 nor R134 at MOI 10 led to tumor growth; the cells
that received the R4 virus grew more slowly than the GVaL-infected cells [Fig.
6(E–G)]. Consistent with the reduced tumor
growth, expressions of both survivin [Fig. 6(I)] and ki67 proteins (a
marker for cell proliferation) [Fig. 6(J)] were significantly repressed
in tumors derived from the cells pre-infected with either R2 at MOI 25 or R4 at
MOI 10. The growth profile of the GVaL-infected SMMC-7721 cells might have
reflected the potential to support tumor expansion that varied with individual
animals. The observation that tumor mass in R134 (100%) by the GVaL infection
was 2-fold bigger than that in R3 (31%) experiments [Fig. 6(K,L)]
favored the conclusion from the previous analyses that three ribozymes acting
together in R134 would have a greater anti-cancer effect than any single
ribozyme (Figs. 3 and 4).To mimic the clinical practice of cancer gene therapy, R3 or R134
viruses were injected in a regime of 3?106 three times every other day into the SMMC-7721
cell-derived tumor (at a volume of approximately 150 mm3) at the
right flank. This resulted in a reduction of tumor growth by 69.5% (R3) and
55.5% (R134) as well as a reduction in tumor volume by 64.8% (R3) and 57.3%
(R134) [Fig. 7(A–C)]. Both survivin and ki67 expression
was significantly reduced in the tumors injected R3 or R134 viruses than the
control viruses [Fig. 7(E,F)].
Discussion
The survivin gene has been regarded as a rational target for
cancer treatment using new molecular antagonists, cancer vaccines and gene
therapeutic agents [3]. Small-molecule antagonists against survivin, including
tetra-O-methyl nordihydroguaiaretic acid to repress Sp1 dependent survivin gene
expression and 17-allyl-amino-geldanamycin or shepherdin to disrupt
survivin/Hsp90 interaction [2326], have entered phase I and II clinical
trials. The use of hammerhead ribozymes seems more advantageous than both RNAi,
because of its target specificity, and antisense, because of its deliverability
by conventional gene therapeutic vectors, including the adenoviral vector used
in this report. The sequence targeted by the R1 in this report is identical to
CUA110, the ribozyme described by Pennati et al. that was also placed
directly within an adenoviral Va I RNA gene in a retroviral vector [25].
However, all characterizations, including cell viability, apoptosis and in
vivo tumor growth, have been performed in stable transformed tumor cell
lines. Choi et al. constructed a hammerhead ribozyme against the same
sequence motif as R3 in this work, where the ribozyme RNA was expressed on its
own in an adenoviral system [14]. Both in vitro and in cell results
demonstrate its ability to cleave survivin RNA as expected and to induce
apoptosis; however, the ability of in vivo to inhibit tumor growth has
not been demonstrated.Here, we show both the specificity and efficacy of each of the four
hammerhead ribozymes to cleave the survivin RNA in vitro (Fig. 3),
in cell (Figs. 4,5), and in vivo (Figs. 6,7). A
combination of three ribozymes (R1, R3 and R4) synergistically suppress
survivin expression [19], which is likely attributable to the fact that there
are three alternately processed survivin transcripts, designated as survivin
(full length), survivin-DEx3 (lacking exon 3) and survivin-2B (retaining part of intron 2 as
a cryptic exon). All forms of survivin RNAs are required for full-blown
apoptosis and aberrant mitosis [20]. A high level of survivin-DEx3 reportedly
correlates with a poorer prognosis in acute myeloid leukemia and cannot be
cleaved by R3 [21], the most potent single ribozyme. Therefore, using R134
virus, which combines three effective ribozymes, should be advantageous, as it
has a higher efficacy at cleaving all the spliced forms of survivin RNA.In addition to the demonstration of the robust therapeutic potential
of hammerhead ribozyme-mediated depletion of survivin expression, this report
has detailed a strategy to develop an effective hammerhead ribozyme that aims
to repress the expression of a given gene, has a rational design that provides
both specificity and efficacy in vitro, in cell culture and in animal,
and includes vectors that will address most, if not all, major technical
concerns.
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