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(1)
(a) In a word, gene therapy and vaccination are two sides of the same adenovirus coin. That is, the gutted adenovirus carries a cargo (foreign gene(s) -- foreign, because the gene(s) does not come adenovirus) which, in both arenas (gene therapy and vaccination) persists in the nucleus of host cells for years, during which the genes (be they foreign and adenoviral, if any) of adenoviral vector are expressed (but the virus do not replicate or multiply).
(b) Now that the introduced gene(s) via adenoviral vector does not integrate into host DNA (and causes disruption of the host DNA), there is no risk of tumorigenesis (cancer-causing) in the host (as there is no way to predict where an introduced gene will integrate into host DNA and what disruption will be). Yet for the same reason, the introduced gene will last for years, not the lifetime of the patient.
(3) Applied Biological Materials (ABM) Inc (2004- ; based in Vancouver, Canada; providing biological service commercially) has an undated Web page titled "Cell Culture - Adenovirus Techniques":
https://old.abmgood.com/marketin ... irus_techniques.php
("Viral vectors are tools commonly used to express exogenous genetic materials in vitro or in vivo. * * * Among them, the most commonly used viral vectors to date are retrovirus, lentivirus, adenovirus and adeno-associated virus (AAV). This section will focus on the recombinant adenovirus expression system. * * * Adenovirus (Ad) was first isolated from adenoid tissue in 1953 * * * [adenoviral] Viral replication and assembly occurs in the nucleus of infected cells and mature viruses leave the host cell via disintegration [of the host]. In contrast to other viruses, adenovirus DNA is not integrated into the host genome and remains in an episomal state [footnote 2 omitted]. Episomes are non-integrated extrachromosomal closed circular DNA molecule that may be replicated in the nucleus [footnote 3 omitted]")
Note:
(a) The adenoid is simply what laypersons call tonsil (more specifically, nasopharyngeal tonsil). There are a pair, which are especially prominent in a child (hence its name 扁桃腺, from its size about an almond -- same as thymus. Which is not surprising, because adenoid and thymus are lymphoid tissue.
(b) There is no need to read the rest of this and the next.
(4) Wang XZ et al, Episomal Segregation of the Adenovirus Enhancer Sequence by Conditional Genome Rearrangement Abrogates Late Viral Gene Expression. Journal of Virology, 74: 11296 (2000)
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC113234/
("Upon introduction into the host cell, the viral chromosome undergoes a site-specific rearrangement to form a circular episome")
The English noun episome was coined by biologists, short from extrachromosomal thing, from Ancient Greek preposition epi on the upper surface of. Wiktionary.com.
(5) Bulcha JT et al, Viral Vector Platforms Within the Gene Therapy Landscape. Signal Transduction and Targeted Therapy, 6: 53 (Feb 8, 2021)/
https://www.nature.com/articles/s41392-021-00487-6
Quote:
"Structure and genome[:]
Ad [adenovirus] is a non-enveloped virus that is known to mostly cause infections of the upper respiratory tract * * * It possesses an icosahedral 二十面体的 protein capsid that accommodates a 26- to 45-kb linear, double-stranded DNA genome. The Ad genome is flanked by hairpin-like inverted terminal repeats (ITRs) that vary in length (30–371 bp [base pairs] at its termini). The ITRs serve as self-priming structures that promote primase-independent DNA replication. A packaging signal located at the left arm of the genome is required for viral genome packaging. The Ad genome encodes ~35 proteins that are expressed in the early and late phases of viral gene transcription. * * *
"Ad as a vector in gene therapy[:]
Ad vectors have the following advantages: (1) high transduction efficiency, both in quiescent and dividing cells [adenovirus enters both non-dividing and dividing cells]; (2) epichromosomal persistence in the host cell; (3) broad tropism for different tissue targets; and (4) and the availability of scalable production systems. Contemporary Ad vectors are derived from human serotypes HAd2 and HAd5 [human adenovirus type 5]. The major objectives in Ad vector development are to overcome the challenges associated with its widely pre-existing viral immunity among the general population, life-threatening strong innate immune responses to its capsid proteins * * *
"The future for Ad vectors[:]
To avoid pre-existing immunity against Ad vectors, several strategies are being employed. First, several 'rare' human serotypes with low seroprevalence, such as HAd2, HAd26, and HAd35, were identified and developed into vectors to minimize pre-existing immunity.
Note:
(a) The senior author and principal investigator of this paper is Guangpin GAO, at University of Massachusetts at medical center in Worcester.
(b) Quotation 1 states, "The Ad genome is flanked by hairpin-like inverted terminal repeats (ITRs) that vary in length (30–371 bp [base pairs] at its termini). The ITRs serve as self-priming structures that promote primase-independent DNA replication."
(i) That means both ends of the one single-stranded DNA off adenovirus double backs, forming a loop on either end. This is expected. Because all replication needs a small segment of nucleotides to continues on; the simile is the phrase: a leg to stand on.
(ii) Okazaki fragments
https://en.wikipedia.org/wiki/Okazaki_fragments
(discovered by Reiji 岡崎 令治 (1930-1975; a victim of Hiroshima atomic 0bombing; died of leukemia) and his wife Tsuneko 恒子 (1933- ) OKAZAKI; section 5 Differences in prokaryotes and eukaryotes)
Virus does not belong to, and is lower in complexity than, a prokaryote.
(c) Johnson & Johnson does not announce the gene structure of its vaccine (trade secret?). So there is no way to comment further.
Johnson and Johnson does say it replies on Janssen's AdVac and PER.C6 cell line. Hence I re-visit
Farson D et al, Development of Novel E1-Complementary Cells for Adenoviral Production Free of Replication-Competent Adenovirus. Molecular Therapy, 14: 305 (2006)
https://www.cell.com/molecular-t ... fulltext/S1525-0016(06)00118-3
("due to the essential role of E1 genes in adenoviral biology, the helper-independent production of E1-modified adenoviral vectors is assisted by cell lines that produce complementary E1 proteins. * * * Expression of E1A in transfected cells can immortalize rodent and human cells. However, expression of E1A promotes apoptosis, which is inhibited by coexpression of E1B. Therefore, cotransfection of E1A and E1B is necessary to establish stable cells that express both E1A and E1B in trans") (footnotes omitted).
(i) Whether used for gene therapy or vaccination, the adenoviral vector contains DNA that does not have E1 region (made up of genes E1A and E1B). Usually E1A and E1B are supplied by a helper virus con-infected with adenovirus. With the PER.C6 cell line, a helper virus is unnecessary.
(ii) This paper said E1A was used to immortalize primary human embryonic retinal cells (or retinoblast). But introduction of the E1A gene alone will kill a cell, so the E1B gene is needed at the same time.
(iii) This is the key: In the absence of E1A and E1B genes, the replication-defective (RD) adenoviral DNA just sits in the nucleus of a host cell (but outside the chromosomes). But to mass produce adenovirus vaccine in a pharmaceutical facility, the RD adenovirus will grow in PERC6, the latter of which produces the complementary E1A and E1B proteins.
(iv) trans-acting
https://en.wikipedia.org/wiki/Trans-acting
(in molecular biology; vs cis-acting)
Trans means different things in chemistry and molecular biology.
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发表于 3-10-2021 11:51:05
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