Hermonat P L, Santin A D, De Greve J, De Rijcke M, Bishop B M, Han L, Mane M, Kokorina N
Department of Obstetrics and Gynecology, University of Arkansas for Medical Sciences, Little Rock 72205, USA.
J Hum Virol. 1999 Nov-Dec;2(6):359-68.
Human adeno-associated virus (AAV) is ubiquitous and known to establish latency by chromosomal integration. We have constructed a wild-type plus AAV vector, ins96-0.9Neo, containing the neomycin resistance gene open reading frame (Neo ORF) of 960 bases in length at map unit 96 of the virus. Ins96-0.9Neo was constructed in an unconventional manner in that the Neo ORF lacked a dedicated heterologous promoter. In this study, this wild-type plus AAV vector was to used to test AAV's packaging capacity and ability for chromosome 19 AAVS1 integration. However, when it was discovered that ins96-0.9Neo also transduced cells to G418 resistance, we also investigated the mechanism of Neo ORF expression in this vector.
STUDY DESIGN/METHODS: We investigated the ability of ins96-0.9Neo to produce virus at high titers and to retain the Neo sequences by Southern blot analysis. The ability of ins96 0.9Neo virus to transduce the Neo gene into cells was analyzed by colony formation under G418 selection, and the ability of ins96-0.9Neo to latently infect cells, including the AAVS1 region of chromosome 19, was investigated by a series of polymerase chain reaction (PCR) amplifications. Finally, the RNA expression of the Neo gene at map unit 96 was investigated by reverse transcriptase primer extension (RTPE) analyses with two different primers and by S1 nuclease protection.
High titers of the ins96-0.9Neo virus could be generated (10(9) infectious units [IU]/mL without concentration), the Neo gene was retained in the encapsidated viral genome, infection by this virus resulted in G418 resistance, and significant integration was taking place within the AAVS1 sequences of human chromosome 19 on transduction. Analysis of mRNA by RTPE using both primers and by the S1 nuclease protection assay mapped the 5' end of the Neo transcripts to approximately 700 bases upstream of the Neo ATG at map unit 81 (nt 3793-3813), thus identifying a new AAV promoter.
These data demonstrate that ins96-0.9Neo will be useful for studying wild-type AAV integration and suggest that such wild-type plus recombinant AAV vectors may be useful for human gene therapy. The advantages of using such wild-type plus AAV vectors over defective AAV vectors include the ease in production of recombinant virus and the ability for site-specific integration into chromosome 19. This study also uncovered a previously unknown AAV promoter, p81. This finding suggests that the as yet uncharacterized ORF (nt 3922-4388) located just downstream of this promoter is likely an expressed gene. Furthermore, these data support our earlier findings that the AAV virion can package >900 bases more than can the wild-type.
人腺相关病毒（AAV）广泛存在，已知可通过染色体整合建立潜伏状态。我们构建了一种野生型加AAV载体ins96 - 0.9Neo，其在病毒图谱单位96处包含长度为960个碱基的新霉素抗性基因开放阅读框（Neo ORF）。Ins96 - 0.9Neo以非常规方式构建，即Neo ORF缺乏专用的异源启动子。在本研究中，该野生型加AAV载体用于测试AAV的包装能力以及整合到19号染色体AAVS1位点的能力。然而，当发现ins96 - 0.9Neo也能将细胞转导为对G418耐药时，我们还研究了该载体中Neo ORF表达的机制。
研究设计/方法：我们通过Southern印迹分析研究了ins96 - 0.9Neo产生高滴度病毒以及保留Neo序列的能力。通过在G418选择下的集落形成分析了ins96 0.9Neo病毒将Neo基因转导到细胞中的能力，并通过一系列聚合酶链反应（PCR）扩增研究了ins96 - 0.9Neo潜伏感染细胞（包括19号染色体的AAVS1区域）的能力。最后，通过使用两种不同引物的逆转录酶引物延伸（RTPE）分析和S1核酸酶保护研究了图谱单位96处Neo基因的RNA表达。
可以产生高滴度的ins96 - 0.9Neo病毒（无需浓缩即可达到10⁹感染单位[IU]/mL），Neo基因保留在衣壳化的病毒基因组中，该病毒感染导致对G418耐药，并且在转导时人19号染色体的AAVS1序列内发生了显著整合。使用两种引物通过RTPE和S1核酸酶保护试验对mRNA进行分析，将Neo转录本的5'端定位到图谱单位81处Neo ATG上游约700个碱基处（nt 3793 - 3813），从而鉴定出一个新的AAV启动子。
这些数据表明ins96 - 0.9Neo将有助于研究野生型AAV整合，并表明这种野生型加重组AAV载体可能对人类基因治疗有用。使用这种野生型加AAV载体相对于缺陷型AAV载体的优势包括重组病毒生产容易以及能够位点特异性整合到19号染色体中。本研究还发现了一个先前未知的AAV启动子p81。这一发现表明位于该启动子下游的尚未表征的开放阅读框（nt 3922 - 4388）可能是一个表达基因。此外，这些数据支持了我们早期的发现，即AAV病毒粒子比野生型能够包装多900多个碱基。
