基因疗法继续承担许多疾病的治疗，但为提交提交有许多和独特的挑战，以作为调查新药（IND）/调查药品掺入（IMPD）用于临床试验的应用。这些应用需要优化的科学计划，旨在解决基因治疗发展的科学，监管和实际挑战。
最近网络研讨会, we discussed gene therapy preclinical areas central to an IND/IMPD submission, namely, pharmacology, toxicology and pharmacokinetics. The following is a brief summary, plus insightful Q&As, of that event.
Evolution of gene therapies
虽然基因疗法在20世纪70年代概念化，但直到20世纪90年代初期，它们成功地用腺苷脱氨酶（ADA）缺乏和家族性高胆固醇血症的治疗方法。不幸的是，千年患有两种临床试验的悲剧 - 其中参与者在逆转录病毒矫正严重联合免疫缺陷（SCID）后开发白血病，以及另一个在腺病毒试验中发生的致命发生以纠正鸟氨酸转基因酰基酶（OTC）。这些不幸的结果导致了交付系统的重新设计，并提高了行业的重点是具有特别强大的科学计划，以确保严格的监管和安全合规性。
What are the challenges associated with IND-enabling programs for gene therapies?
Gene therapies require tailored toxicology
Current Guidelines and evolving regulatory standards within preclinical studies can be an issue; although, a multi-disciplinary approach is pertinent in addressing to the toxicology/pharmacology of each specific evaluated therapy. Routes of exposure (e.g. intrathecal or intravenous) and pharmacokinetics (e.g. duration or concentration of exposure) will help reveal potential adverse effects. Usually, most standard approaches cannot be applied to these precision medicines – for example, gene therapy generally utilizes a single species evaluation versus the conventional two.
Each gene therapy requires a unique regulatory approach
从监管角度来看，建议使用美国食品和药物管理局（美国FDA）和在治疗发展过程中的其他监管机构。此外，必须提交化学，制造和控制（CMC）信息（超出该特定网络研讨会和博客的范围的区域），以确保调查产品的一致产品安全，身份，纯度和效力。
Programs may encompass improving upon a current approved therapy or addressing an unmet need in a therapeutic indication. As there is no ‘one-size-fits-all’, working closely with regulators is essential when designing and progressing toward clinical trials. The INitial Targeted Engagement for Regulatory Advice on CBER (Center for Biologics Evaluation and Research) producTs (INTERACT) meetings at the US FDA facilitate identification of issues at an early stage; many public facing regulatory bodies have established and conduct similar programmatic meetings across the globe. This approach is especially useful in dealing with innovative investigational products with unique safety profiles. Fast track development paths within the US FDA are also of paramount importance, as they can offer expedited review and approvals when preliminary evidence suggests that the product benefits are greater than those currently available.
In vivo model selection can impact outcomes
存在多种模型选项，并且物种选择取决于解剖学，疾病模型和相关的interspecies信号通路。最常见的是使用非患病的动物模型。然而，在不了解真正的组织表达谱的情况下，正常的健康动物可能会遇到某些疗法过度表达的问题。因此，有两个推荐方法可以说明临床前计划内的功效和相关安全性。yaboapp体育官网简而言之，其中的第一个仅使用疾病模型;第二种是使用健康和患病动物的混合安全设计，当患病模型可能具有增强早期发病率的潜在的健康问题时，允许设计中的控制元素。在部署病毒递送基因疗法时，还需要考虑免疫抑制方案，其中成功的结果是基于科学工作人员的专业知识和动物的特殊处理和/或住房。
Accounting for past exposure to viral vectors
对腺相关载体（AAV）构建体的预先存在的抗体的测试对于理解引发免疫原性反应，降低转导率或可能致命反应的可能性至关重要。动物需要至少预先筛选两次：早期筛查，以确保它们对过去暴露的负面影响;和第二次筛选，就在给药之前，以确保血管转换。应考虑使用血清或脑脊髓液（CSF）的互补测定和细胞因子测定。慢病毒（Lenti）构建体通常不需要这些类型的预先存在的抗体评估。
Taking precautions against viral shedding
Certain aspects of preclinical study designs need to be considered when utilizing viral elements of delivery. One of these pertains to the timing of dose administration. When cohort dosing or split initiation is separated by more than 2 to 3 days, the dosed and non-dosed animals need to be housed separately to avoid cross contamination of shed (excreted) viral particles via, for example, feces, urine or saliva which may impact to results interpretation. In addition, appropriate personal protective equipment (PPE) for technicians and appropriate hygiene practices for room entry and egress should be employed to ensure the safety of the technical staff and to prevent cross contamination between animals and rooms.
确定基因治疗的最佳施用途径
As well as the traditional systemic routes of administration, gene therapies are frequently administered via the CNS or ocular system for a more targeted delivery, depending on the specific disease indication being considered for treatment.
Ocular administration
在Covance,我们注入的50 - 100毫升左右viral vector by intravitreal, subretinal or suprachoroidal routes. These injections can expose the subject to smaller viral loads of around 108–109拷贝/毫升作为目标是inj近端ection. Whilst leakage from the site of injection can occur, e.g. into the optic nerve, ocular delivery can be optimised, and risks minimized, by working with specialised veterinarian ophthalmologists. Further, we partner with Ocular Services On Demand (OSOD)– a world-class team of vision scientists to ensure exceptional study design, execution and results interpretation. Functional changes in the reactivity of the eye can be assessed during the live phase and specialized tissue collections post-life can evaluate the biodistribution within the eye as well as assess any changes in histology.
中枢神经系统（CNS）管理
CNS管理利用CSF方便和逆行流动用于病毒载体分布。更容易且临床相关的接入点位于低于Conus Medullaris水平的腰椎内，而是在Cauda Equina的区域内。将钝针插入弯曲脊柱的椎骨（第三腰椎）之间的腰部鞘内空间中。除去脑脊髓液，在至少1分钟的过程中轻轻注入相同的试验材料制剂的剂量。一旦针被移除，将动物置于Trendelenburg位置以进行改进的分布。其他路线包括注入岩壁麦塞卡，将材料放置在第四脑室和中央管接近的材料，并注射靶向侧脑室的脑室内部区域，以及使用立体定位MRI引导剂量给药的特异性实质区域以确保靶向交货。后剂量临床观察应包括评估疼痛或痛苦（头痛）和由于注射压力增加而潜在的神经缺陷;关于后者，分析注射率和体积对于最小化影响至关重要。
Designing biodistribution assessments
Biodistribution needs to be monitored at intervals generally ranging from 1–12 months and is key to understanding the distribution of product in multiple tissue types. Even with targeted delivery, viral particles can become widely distributed; thus multiple systemic tissues should also be evaluated. The gonads should also be analyzed as concentration here may influence potential transfer to offspring. In the brain (flash-frozen or fixed), the right side is often used for biodistribution and the left side for histology. There is also a known risk of severe toxicity and degeneration of the dorsal route ganglia (DRGs) after viral vector therapy. It is a labour-intensive task to analyze the regions within the DRG, for example: the cervical, thoracic and lumbar regions, however these can be collected and analyzed for transgene product as well as histopathology. Distal peripheral nerves as well as ascending tracts within the spinal cord should also be evaluated histologically to determine extent of changes.
结论
在Covance，我们简化了开发独特的精密药品的旅程。我们在基因治疗空间中展示了专业知识，帮助科学家克服了毒理学，CMC测试，生物分析分析，生物标志物和迅速过渡到人类试验中的挑战和研究设计考虑因素。
Webinar Q&A
问：您是否可以评论基于预筛选抗体滴度的包含和排除标准，并且通常用于中和抗体的测定？
答：大多数人和动物测试受试者经历了一些暴露于腺相关病毒（AAV）的特定血清型。在测试动物中，我们的目标是≤1：10滴度。重要的是要理解预筛查协议以及实验室定义为“消极背景”以及如何相关的实验室。有时我们必须利用血清阳性动物作为对照，因为特定AAV血清型的电流供应中可能没有足够的血清可动物。至关重要的是了解测定的截止和每只动物的情况。
Q: Can you control the copy number? What range of copy number is typical?
A: Copy number is dependent on dose and route of administration, although 108–1014是典型的，眼睛或大脑108通常是足够的。较高的拷贝数通常等同于病毒的更大生物分布。
问：Cocance如何解决感染性测定中的样品？
答：我们进行的大多数测定都表现出阳性或负面脱落，而不是评估这些样品的感染性。非团体样品（例如粪便，尿液等）是挑战，而且in vitro斑块测定形成特别难以保持清洁。
Q: What is the stance at Covance on readouts for biodistribution – DNA, RNA or protein? Or a combination?
A: Typically, readouts for gene therapy biodistribution has used DNA, RNA or protein – or the activity of that protein. DNA via qPCR is the most common method. However, in consideration of RNA and protein, it may be difficult to discern between inter-species RNA and establish if the measured activity results from, for example, the humanized recombinant versus native species enzyme. To differentiate between the two isoforms, an examination of the mRNA indicates that the target gene is being transcribed. It is vital to differentiate between endogenous protein and exogenous product using techniques like electroretinography (ERG), electroencephalogram (EEG) and Doppler to evaluate changes in targeted function if possible.
In some programs, we are able to identify the DNA within tissue, but not the RNA or protein, so establishing an argument on the mRNA or protein readout will help convince regulatory bodies that your product is likely to function correctly.
Q: What is your opinion on tissue distribution?
A: It is important to be as comprehensive as possible. We recommend collecting approximately 14-20 tissues at necropsy. For example, if one is administering viral vectors into the brain, then samples of tissues from the brain, spinal column or spinal cord are clearly required. It’s important to collect and analyze tissues that may experience systemic exposure such as the liver, kidneys, intestine, eye and draining lymph nodes. Also it is paramount to check distribution and/or expression in the gonads – as questions related to germline cell transfer will allow one to be prepared to address that concern.
Q: What PK time points do you recommend for AAV IM or IV routes of administration?
答：给定IGM波通常在前6-8天后增加，等同于AAV载体的快速下降，IgG波反应在10天内产生循环AAV的全部间隙;寻址病毒衣壳的PK更像是疫苗接种反应。随着AAV和用于提供基因疗法的大多数病毒是非复制的，曾经施用则没有持续的膨胀病毒生产。此属性将降低产品的表达，然而，生物分布将持续更长的时间。我们不建议在当天进行抽样，因为病毒需要进行细胞和表达需要建立。然而，在第一周之后可以收集时间点，然后在每周或每月间隔随访，以进行研究持续时间，并且是特异性的治疗，特别是如果转基因产生进入循环的产品
Abbreviations
AAV adeno-associated virus
ACO反义寡核苷酸
ADA adenosine deaminase
CBER Center for Biologics Evaluation and Research
CMC化学，制造和控制
CSF脑脊髓液
DNA deoxyribonucleic acid
DRG dorsal route ganglia
FDA.Food and Drug Administration
Ind Investigational新药物
互动初步有针对性的参与CBER（生物学评估和研究中心）产品（互动）
MRI magnetic resonance imaging
OSOD Ocular Services On Demand
OTC OTNITHINE TRANSCARBAMYLBAMYL
RNA ribonucleic acid
SCID severe combined immunodeficiency
