E4腺病毒基因对病毒与细胞启动子影响研究

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"这篇文章是关于E4腺病毒基因对病毒和细胞启动子影响的研究论文，由Linda Grave等人撰写，并在2000年发表。研究团队来自Transgene SA和Crucell BV，探讨了E4基因如何在体内外影响不同启动子的活性，对于理解重组腺病毒载体在人类基因治疗中的应用具有重要意义。" 文章详细内容: 腺病毒基因E4在基因治疗领域扮演着关键角色，因为它能显著影响病毒和宿主细胞的转录过程。E4基因区域包含了多个开放阅读框（ORFs），这些ORFs编码的蛋白质参与病毒生命周期的多个阶段，包括DNA复制、宿主细胞周期调控以及免疫逃避等。本研究旨在深入理解E4基因如何具体地调节病毒和细胞的启动子活性，这对于优化重组腺病毒载体的性能至关重要。研究人员构建了一对同源E1缺失的E1u和E1uE4u病毒载体，以比较E4基因存在与否对转基因表达的影响。通过比较这两组载体在体外细胞培养和体内实验模型中的表现，他们评估了各种启动子的活性变化。这可能涉及到E4基因产物对启动子的直接结合或通过细胞内信号通路间接影响。实验结果可能揭示了E4基因在某些情况下增强或抑制转录的具体机制，这将有助于设计更有效的基因疗法策略。例如，如果E4基因能够增强特定启动子的活性，那么这种增强可能被利用来提高治疗基因在目标细胞中的表达水平。反之，如果E4基因抑制了某些启动子，那么避免或阻断这种效应可能成为减少副作用的手段。此外，E4基因对细胞启动子的影响可能与它在调节宿主细胞周期和免疫反应中的作用有关。这些发现可能解释了为什么在某些基因治疗临床试验中，尽管E4基因被删除或失活，但仍观察到不理想的转基因表达模式。理解这些复杂的相互作用可以指导科学家们开发出更安全、更高效的病毒载体，以传递治疗基因至患者体内。 Linda Grave等人进行的这项研究加深了我们对E4腺病毒基因作用的理解，为改善基因治疗工具的设计提供了宝贵的理论依据。通过进一步的研究，这些发现可能会促进新型基因疗法的发展，从而更好地服务于遗传疾病和慢性病的治疗。

(Swiss-3T3, embryonic ®broblast; NIH-3T3, embryon

contact-inhibited ®broblast; Mus Dunni, normal tail

®broblast) and canine (MDCK, kidney epithelioid) cell

lines were obtained from the ATCC. The human 293-

E4ORF6/7, simian W162 (Ad5 E4 transfected Vero),

canine CML10 (melanoma) and feline PG4 (brain

astrocytes) cells were as previously described [24,67±69].

Animal studies

CB17-scid/scid immunode®cient mice and immunocom-

petent B6D2 (H-2

bxd

) mice (6±8 weeks old) were

purchased from IFFA-CREDO (L'Arbreles, France). The

viral vectors were administered at the indicated doses by

intra-tracheal (it), intravenous (iv) or intra-tumoral (itu)

injections. All vectors were diluted either in the viral

storage buffer (iv, itu) or in 0.9% NaCl (it). Blood samples

were collected at different times post-injection from the

mice injected iv or it and were conserved at x20uC until

analysis. Animals were killed 3 days after itu injection for

tumor extraction; tumors were crushed in PBS1X and

extracts were stocked at ±20uC prior to analysis.

DNA analysis

Total DNA was extracted from tissue culture cells and

organs as described previously [24]. Brie¯y, cells or

tissues were digested overnight with a proteinase K

solution (1 mg proteinase K in 1% SDS) in DNA lysis

buffer (10 mM Tris-HCl pH 7.4, 400 mM NaCl, 2 mM

EDTA). Total cellular DNA was isolated by phenol-

chloroform extraction followed by ethanol precipitation.

DNA (5±10 mg) was digested with the following restric-

tion enzymes: Nco1 for the hIL2 Ad vectors and BamH1

for the hCFTR Ad vectors. The quality and amount of DNA

were monitored by ethidium bromide staining of the gels

prior to transfer. Analyses by Southern blot were

performed as described elsewhere [70], using appropriate

P-labeled cDNA probes: for the hCFTR Ad genomes, the

probe detected a 2540 bp hCFTR-internal fragment. This

fragment does not include the intron. For the hIL2 Ad

genomes containing the chimeric intron in the expression

cassette, the probe detected a fragment of 1687 bp; for

the hIL2 Ad genomes without the chimeric intron in the

expression cassette the probe detected a 1443 bp frag-

ment.

RNA analysis

The steady-state level of the hIL2 and hCFTR mRNAs was

monitored by Northern blot analysis. Total RNA was

extracted from tissue culture cells using the RNA Now kit

(Ozyme, Saint-Quentin-les-Yvelines, France) according to

the manufacturer's instructions. For Northern blot

analysis, 10 mg total RNA was subjected to agarose gel

electrophoresis [70] and transferred to nitrocellulose

®lters. Filters were stained after transfer to ensure that

equal amounts of total cellular RNA were loaded and

transferred. hIL2, hFIX and hCFTR mRNAs were then

detected by hybridization using appropriate

P-labeled

DNA probes (corresponding cDNAs).

ELISA assays

The concentration of the secreted transgene products

(hIL2 or hFIX) was determined from cell culture super-

natants, mouse sera or tumor extracts by the following

enzyme-linked immunoadsorbant assays (ELISA) accord-

ing to the manufacturer's protocol: Quantikine hIL2 (R&D

Systems, Oxfordshire, UK) and Asserochrom IX: Ag kit

(Diagnostica Stago, Asnie

re-sur-Seine, France).

Nuclear run-on transcription assay

Methods for the analysis of the labeled nascent RNA were

essentially as described previously [71]. Brie¯y, A549 or

Vero cells were transduced at a multiplicity of infection

(MOI) of 100 IU/cell with the indicated vectors. Nuclei

were isolated 48 h post-transduction and incubated in

vitro to allow previously initiated nascent transcripts to be

elongated by polymerases. To this end, cell culture-

initiated RNA transcripts from 2r10

nuclei were

elongated for 30 min at 30uC in the presence of 200 mCi

[a-

P]UTP (3000 Ci/mmol) in a ®nal volume of 200 ml

containing 1 mg/ml heparin, 0.6% (vol/vol) sarkosyl,

0.4 mM concentrations of ATP, GTP, CTP, 2.5 mM

dithiothreitol, 0.15 mM phenylmethylsulfonyl ¯uoride

(PMSF) and 350 mM (NH

)

. Termination of the

reaction and isolation of labeled RNA was exactly as

described previously [71]. Dried RNA pellets were

dissolved in hybridization buffer containing 40% for-

mamide, 2 mM EDTA, 0.9 M NaCl, 50 mM Na

HPO

NaH

, pH 6.5, 1% SDS, 0.4 g/l polyvinylpyrrolidone,

0.4 g/l Ficoll, 50 g/l dextran sulfate and 50 mg/l dena-

tured salmon sperm DNA. Labeled RNA from infected

A549 or Vero cells (7r10

and 7r10

cpm, respectively)

were used to hybridize to linearized plasmid DNA

containing the hCFTR cDNA, the hb-actin cDNA (internal

control) and the plasmid backbone (negative control),

previously denatured in the presence of 0.3 M NaOH and

immobilized on a nitrocellulose membrane (5 mg plasmid

DNA/dot). Prehybridization (18 h) and hybridization (3

days) were carried out at 42uC in the same buffer (see

above). Filters were washed twice in 2rSSC, 1% SDS for

15 min at 22uC and twice in 0.1rSSC, 0.1% SDS for

15 min at 52uC. Radioactivity bound to the ®lters was

quanti®ed by scintillation counting.

FACS scan analysis

The levels of eGFP expression in transduced cells were

detected and quanti®ed by ¯ow cytometric analysis

(FACS Scann, Becton Dickinson, Grenoble, France).

Brie¯y, cells were washed and resuspended in PBS,

¯uorescence analysis was performed using 10

cells and

the mean of ¯uorescence was measured in comparison to

untreated cells.

Role of E4 on Transgene Expression 435

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E4腺病毒基因对病毒与细胞启动子影响研究

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