Investigation into factors controlling the efficacy of integration deficient lentiviral vectors for gene delivery

载体或基因传递载体已经发展到将DNA和基因转移到细胞中。载体是研究基础生物学的重要工具，包括基因如何起作用、细胞如何工作以及控制动物发育过程的因素。许多疾病是由有缺陷的基因引起的，所以最近载体也被开发用于基因治疗；用基因的功能拷贝来修复缺陷。最有效的载体是从病毒中发展出来的，这些病毒经过数百万年的进化，可以将遗传物质传递到细胞中。为了使病毒成为安全的工具或药物，病毒的致病性已经被去除，这样病毒就可以在实验室中生产，携带特定的感兴趣的基因。人类免疫缺陷病毒（HIV）是一种安全的慢病毒，是一种常用的将基因传递到细胞的载体。该载体非常有效，已用于临床基因治疗试验。当HIV通常感染目标细胞时，它会将其遗传载荷直接传递到宿主细胞染色体中，并将其无缝整合，从而使病毒DNA成为细胞基因组或“生命之书”的一部分。这对长期实验或治疗非常重要，因为DNA是永久传递的。然而，尽管载体不能引起感染，但将基因整合到宿主细胞的染色体中存在问题和副作用。并不是所有的细胞DNA都是活跃的；基因组中有很大一部分不能从它们所包含的信息中产生任何产物。如果病毒或载体将其有效载荷插入其中一个区域，它发挥作用的机会就会降低。此外，细胞在数百万年的时间里也发生了变化和进化，以抵御病毒（或由病毒衍生的载体）传递给它们的DNA。目前存在的机制可以关闭传递基因的生产（或表达）并使其沉默。最后，将DNA插入细胞的一个副作用是，它会干扰细胞自身的DNA，损害或改变现有的基因。这不大可能引起任何问题，但如果在治疗患有遗传性疾病的人时，它会影响控制细胞生长或复制方式的基因，那么这可能会导致癌症。虽然这是一个非常小的风险，但不幸的是，在基因治疗临床试验中观察到它。另一种方法是使用经过改变的HIV载体，以防止其将携带的DNA插入目标细胞染色体。这些载体被称为整合缺陷慢病毒载体或idlv。这些载体不再能够整合到细胞的基因组中，但这个载体保留了整合版本的所有其他好处。这有几个后果：首先，它应该更安全。由于传递的DNA没有整合，它不会破坏细胞自身的任何基因，这降低了患者患癌症的风险。有趣的是，尽管避免了与基因组沉默区域整合相关的问题，但与整合载体版本相比，IDLV在所有细胞类型中并不同样有效，原因尚不清楚。虽然它们已被证明可以有效地表达大脑、眼睛和肌肉组织中的基因，但与整合载体相比，它们在肝脏和干细胞中的表现更差。这种阻滞可能是由许多因素造成的，但由于idlv代表了一种更安全、可行的替代方案，因此研究其原因以找到解决方案或确保所传递基因的表达最终不会在它们最初似乎工作良好的组织中受到影响是很重要的。因此，本项目的目的是确定影响整合缺陷慢病毒载体在不同情况下如何发挥作用的因素，并利用该信息产生改进的载体。

Vectors or gene delivery vehicles, have been developed to transfer DNA and genes into cells. Vectors are important tools which have been used to investigate basic biology, including how genes function, how cells work and what controls the process of animal development. Many diseases are caused by faulty genes and so recently vectors have also been developed for use in gene therapy; where a functional copy of a gene is used to fix the defect. The most effective vectors have been developed from viruses which have evolved over millions of years to deliver genetic material into cells. To make viruses into safe tools, or medicines, the pathogenic properties of the virus have been removed so the virus can be produced simply in a laboratory for carrying specific genes of interest. The Human Immunodeficiency Virus (HIV) is a lentivirus that has been made safe and is a commonly used vector for delivering genes to cells. This vector is very effective and has been used in clinical gene therapy trials. When HIV normally infects a target cell, it delivers its genetic payload directly into the host cell chromosomes and integrates it seamlessly, so that the viral DNA becomes part of the cell's genome or 'book of life'. This is very important for long-term experiments or treatments, because the DNA is delivered permanently. However, despite the fact that vectors are unable to cause infection, there are problems and side effects associated with integrating genes into the host cell's chromosomes. Not all of a cell's DNA is active; there are large regions of the genome that do not produce any product from the information that they contain. If the virus, or vector, inserts its payload into one of these regions, the chances of it functioning are reduced. Additionally, cells have also changed and evolved over millions of years to defend against DNA that is being delivered into them by viruses (or vectors derived from them). Mechanisms are present that can shut off production (or expression) from delivered genes and silence them. Lastly, a side effect of inserting DNA into a cell is that it can interfere with cell's own DNA and damage or alter existing genes. This is unlikely to cause any problems but if, when treating a person with an inherited disease, it affects a gene involved in controlling how a cell grows or replicates, then this can lead to cancer. Although this is a very small risk, it has unfortunately been observed in gene therapy clinical trials. An alternative is to use an HIV vector that has been altered to prevent it from inserting the DNA it is carrying into the target cell chromosome. These vectors are called integration deficient lentiviral vectors or IDLVs. These are no longer capable of integrating into the cell's genome, but this vector retains all other benefits of the integrating version. This has several consequences: Firstly, it should be safer. Because the delivered DNA does not integrate, it cannot disrupt any of the cell's own genes and this reduces the risk of causing cancer in patients. Interestingly, despite avoiding the problems associated with integrating into silent areas of the genome, IDLV do not work equally well in all cell types when compared to integrating versions of the vector, for reasons that are not clear. While they have been shown to effectively express genes in brain, eye and muscle tissue, they perform worse in liver and stem cells when compared to integrating vectors. This block could be due to many factors but because IDLVs represent a safer and viable alternative to those that integrate, it is important to investigate the reasons to find solutions or to ensure that expression of delivered genes is not eventually affected in tissues where they initially seem to work well. The aim of this project therefore is to determine factors that influence how integration defective lentiviral vectors function in different situations and use that information to produce an improved vector.