The progress from clinical development toward product licensure of several programs presents challenges mainly to gene therapy product manufacturing. These include challenges in viral vector‐manufacturing capacity but also in process variability, difficulty characterizing complex materials, and lack of knowledge of critical process parameters and their effect on critical quality attributes of the viral vector products. In the viral vector production, scale‐up is a critical task due to the limited scalability of traditional laboratory systems and the demand for high volumes of viral vector manufactured following current good manufacturing practice.

Furthermore, the expanding potential commercial product pipeline in the last years and the continuously advancing development of viral vectors for gene therapy require that products are well characterized and consistently manufactured to rigorous tolerances of purity, potency, and safety. Finally, there is an increase in regulatory scrutiny that involves also the academic researchers and manufacturers of investigational drugs for early‐phase clinical trials engaged in industry collaborations.

This trend has led to a greater demand for both pre‐clinical and clinical‐grade viral vector‐manufacturing capacity to support the increasing number of gene therapy clinical development programs. As these programs advance toward licensure, more rigorous product characterization using improved analytical methods and progressively higher regulatory compliance will be expected.

The solution to increasing viral vector capacity involves both scientific and technical issues. The manufacturing should be robust, scalable, and more automated to fit GMP solutions for both small‐scale and large‐scale production. In clinical manufacturing, procedures must produce consistent results with a minimum lot to lot variability. For example, variability in transfection efficiency and the lack of scalability and consistency is therefore problematic and requires rigorous control. Ferreira et al.^[1] review recent progress in stable cell line development to overcome this challenge in the manufacturing of lentiviral vectors (LV). Similar developments are performed for the production of AAV vectors (development of stable virus‐free inducible AAV producer cell lines based on CAP cells^[2] and in the future in the context of the baculovirus/insect cell system.^[3] Alternatively, Lesch et al.^[4] evaluate the use of single‐use fixed‐bed bioreactors for large‐scale production of viral vectors (AAV, LV, retroviral, and adenoviral vectors) as well as viruses for vaccine purposes. Further improvements in the reactor production are achieved by the establishment of high cell density culture systems as shown for the baculovirus/Sf9 system for the production of AAV under optimized conditions (10 × more cells produce also ten times more vector amount)^[3] or for MVA production under perfusion conditions (Gränicher et al.^[5]—comparison of different perfusion systems).

Though reactor systems have been improved and will be further improved for the increasing vector productivities of any vector system, it is obvious that the biological system is the base and its optimization will be even more important than the reactor‐based production system. This concerns any biological system and in the context of this special issue, as an example, Jacob et al.^[6] present an improvement of the baculovirus system for AAV production (concerning passage stability and safety) by replacing the traditional Tn7 transposition with straightforward homologous recombination.

Purification could be another bottleneck of viral vector manufacturing since most of the available technologies tend not to fit the desired needs. Downstream processes for viral vectors are far from being mature, as new resins, matrices, and operation modes are being developed aiming for better purification processes with high vector yields, reducing overall purification cost and time. Moreira et al.^[7] review recent progress in the purification of lentiviral vectors. El Andari and Grimm‘s^[8] paper present in their review methods for the purification of different serotypes of AAV vectors with special emphasis on synthetic AAV gene therapy vectors (AAV‐DJ and ancAAV). Furthermore, Dickerson et al.^[9] report a new method for separating empty and full rAAV particles—a highly important issue when moving to whole‐body gene therapy treatment because empty AAV particles can be considered as a contaminant—using isocratic anion exchange chromatography that could be used with minor changes for different serotypes.

Today one of the most important vectors in the in vivo gene therapy field is the rAAV. Using this vector system, for whole‐body treatments, such as the treatment of DMD or XLMTM (X‐linked Myotubular Myopathy) patients, huge vector amounts are required (e.g., 1–3 × 10¹⁴ vg kg^‐1 (Gene Transfer Clinical Study in X‐LMTM (ASPIRO), NCT03199469). This signifies that on one hand, the production system must be highly efficient in view of the production of the required vector amounts (see above) and that on the other hand, the purification protocol must be able to remove any contaminant, including empty AAV capsids, to avoid/preclude (severe) adverse events (see above).

This requires, in particular, viral vectors destined for in vivo use, such as AAV vectors, the availability of efficient monitoring methods during process development and proper characterization, and robust quantification methods in the phase of routine manufacturing to cope with regulatory demands. In the context of process development, papers by Wright,^[10] Lecomte et al.,^[11] and El Andari and Grimm^[8] present different aspects of analytical methods required for the deep characterization of rAAV vectors for R&D as well as for process developmental purposes, including, for instance, the development of an improved method for high throughput sequencing to identify and quantify DNA species in recombinant AAV batches,^[11] mass spectrometry‐based methods to characterize AAV capsid protein integrity,^[8] vp1,2,3 stoichiometry,^[8] deamidation^{[10, 12]} and oxidation^{[10, 13]}/phosphorylation^[2] or differential scanning fluorimetry to measure AAV capsid thermostability.^[8] Furthermore, the paper by Wright^[10] reviews quality control methods for routine testing of AAV vectors.

Finally, the paper by Romito et al.^[14] presents a viral vector‐free ex vivo gene therapy approach based on the use of electroporation in view of gene editing of primary CD4+ T cells for rendering them resistant to HIV‐1 infection (disruption of CCR5 alleles). In addition to the stable modification, the absence of mutagenic events was shown. The interesting aspect of this approach is that in the absence of the use of viral vectors, no insertional mutagenesis is to be expected.

This special issue can only provide a relatively limited glimpse of the actual state of gene therapy/gene modification. Despite the tragic deaths observed during the ASPIRO clinical trial,^[15] in vivo gene therapy will continue to evolve by using improved and safer rAAV vectors and in future other viral vectors and, in particular, lentiviral vectors.^[10] On another side, it can be expected that in the context of ex vivo gene therapy, non‐viral gene therapy/genome editing^[14] will become a competitor to the use of retro/lentiviral vectors. However, the focus is on the cure and well‐being of patients, meaning that only the most advanced and safest treatment must be applied.

Cristina Peixoto

Otto‐Wilhelm Merten

从临床开发到几个程序的产品许可的进步提出了主要对基因疗法产品制造的挑战。这些挑战包括病毒载体生产能力方面的挑战，以及过程变异性，复杂材料的表征困难，对关键过程参数的了解及其对病毒载体产品的关键质量属性的影响等。在病毒载体的生产中，由于传统实验室系统的可扩展性有限，并且需要按照当前良好的生产规范生产大量病毒载体，因此扩大规模是一项至关重要的任务。

此外，最近几年潜在的商业产品渠道不断扩大，以及用于基因治疗的病毒载体的不断发展，要求对产品进行良好的表征并始终如一地制造，以达到严格的纯度，效力和安全性要求。最后，监管审查的增加也涉及从事行业合作的早期临床试验的学术研究人员和研究药物的生产商。

这种趋势导致对临床前和临床级病毒载体生产能力的需求增加，以支持越来越多的基因治疗临床开发计划。随着这些计划朝着许可的方向发展，使用改进的分析方法以及逐步提高的法规遵从性将要求更严格的产品表征。

增加病毒载体能力的解决方案涉及科学和技术问题。制造应具有健壮性，可扩展性和更高的自动化程度，以适合小规模和大规模生产的GMP解决方案。在临床生产中，程序必须以最小的批次间差异产生一致的结果。例如，因此，转染效率的可变性以及可伸缩性和一致性的缺乏是有问题的，并且需要严格的控制。费雷拉等。^{[ 1 ]} 综述稳定细胞系开发的最新进展，以克服慢病毒载体（LV）生产中的这一挑战。AAV载体的生产也进行了类似的开发（基于CAP细胞^{[ 2 ]}以及将来在杆状病毒/昆虫细胞系统中的稳定无病毒诱导型AAV生产者细胞系的开发。^{[ 3 ]}或者，Lesch等^{[ 4 ]} 评估一次性使用固定床生物反应器大规模生产病毒载体（AAV，LV，逆转录病毒和腺病毒载体）以及用于疫苗的病毒的情况。通过建立高细胞密度培养系统（如杆状病毒/ Sf9系统所示）在优化条件下生产AAV可以进一步提高反应器产量（10倍以上的细胞也产生十倍的载体量）^{[ 3 ]}或在灌流条件下生产MVA（Gränicher等人^{[ 5 ]} –不同灌流系统的比较）。

尽管为提高任何载体系统的载体生产率而对反应器系统进行了改进并将进一步加以改进，但显然生物系统是基础，其优化比基于反应器的生产系统更为重要。这涉及到任何生物系统，在本期特刊中，例如Jacob等。^{[ 6 ]} 通过用简单的同源重组代替传统的Tn7转座，提出了一种用于AAV生产的杆状病毒系统的改进（关于传代稳定性和安全性）。

纯化可能是病毒载体生产的另一个瓶颈，因为大多数可用技术都无法满足所需的需求。病毒载体的下游工艺还远远不成熟，因为正在开发新的树脂，基质和操作模式，目的是为了以更高的载体产量实现更好的纯化工艺，从而减少总体纯化成本和时间。Moreira等。^{[ 7 ]} 综述了慢病毒载体纯化的最新进展。El Andari和Grimm ^{[ 8 ]}在他们的综述方法中提出了不同血清型AAV载体的纯化方法，特别侧重于合成AAV基因治疗载体（AAV-DJ和ancAAV）。此外，迪克森等。^{[ 9]} 报告了一种新的分离空和完整rAAV颗粒的方法-当转向全身基因治疗时，这是一个非常重要的问题，因为空的AAV颗粒可以被认为是一种污染物-使用等度阴离子交换色谱法，可以对其进行较小的改动不同的血清型。

如今，体内基因治疗领域最重要的载体之一是rAAV。使用这种载体系统进行全身治疗，例如DMD或XLMTM（X连锁肌管肌病）患者的治疗，需要大量的载体（例如1-3×10 ¹⁴ vg kg ^-1（基因转移临床在X‐LMTM（ASPIRO）中进行的研究（NCT03199469），这表明，一方面，鉴于需要的载体量的产生，生产系统必须高效（见上文）；另一方面，纯化方案必须高效必须能够清除任何污染物，包括空的AAV衣壳，以避免/避免（严重）不良事件（请参阅上文）。

这特别需要用于体内使用的病毒载体，例如AAV载体，在工艺开发过程中进行有效监控的方法和适当的表征方法，以及在常规生产阶段应对法规要求的可靠的定量方法。在过程开发的背景下，Wright ^{[ 10 ]} Lecomte等^{[ 11 ]} 以及El Andari和Grimm ^{[ 8 ]的论文}介绍了用于研发和过程开发目的的rAAV载体的深度表征所需的分析方法的不同方面，例如，包括改进的高通量测序方法的开发，以鉴定和定量重组AAV批次中的DNA物种，^{[ 11 ]}基于质谱的方法来表征AAV衣壳蛋白完整性，^{[ 8 ]} vp1,2,3化学计量，^{[ 8 ]}脱酰胺作用^{[ 10,12 ]}和氧化^{[ 10，13 ]} /磷酸化^{[ 2 ]}或差示扫描荧光法测量AAV衣壳的热稳定性。^{[ 8 ]}此外，Wright ^{[ 10 ]}的论文回顾了AAV载体常规检测的质量控制方法。

最后，Romito等人的论文。^{[ 14 ]} 提出了一种基于电穿孔的无病毒载体离体基因治疗方法，以期对原代CD4 + T细胞进行基因编辑以使其具有抗HIV-1感染（CCR5等位基因破坏）的能力。除了稳定的修饰外，还显示出没有诱变事件。该方法有趣的方面是，在不使用病毒载体的情况下，预期不会发生插入诱变。

这个特刊只能让人们相对有限地了解基因治疗/基因修饰的实际状态。尽管在ASPIRO临床试验中观察到悲剧性死亡，^{[ 15 ]}体内基因治疗将继续通过使用改良和更安全的rAAV载体以及未来的其他病毒载体，尤其是慢病毒载体来发展。^{[ 10 ]}另一方面，可以预期在离体基因治疗的背景下，非病毒基因治疗/基因组编辑^{[ 14 ]}将成为使用逆转录/慢病毒载体的竞争者。但是，重点在于患者的治愈和健康，这意味着仅必须采用最先进，最安全的治疗方法。

克里斯蒂娜·佩索托（Cristina Peixoto）

奥托·威廉·默滕