Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_5943_Библиотеки_им_академика_М_И_Перельмана

the same delivery system/procedure as planned for the clinical study should be feasible, which may not always be the case in small animals like mice. The selected species should reflect the patient population regarding disease‐specific physiolog­ical conditions (e.g. blood‐ocular barrier could be impaired in patients), sex, and age and is biologically responsive to the expressed therapeutic transgene[1]. It also needs to be considered that, preexisting immunity as well as immune responses mounted to the administered GT product and its expression products may affect the BD profile of the vector and the transgene expression product(s) and may be associated with adverse events (e.g. liver enzyme increase) [15]. Nonhuman primates may be the best choice to assess immune‐related effects on BD or to evaluate new regulatory elements, whereas disease models in most cases will be only available in mice. In vitro studies comparing transduction efficiency and transgene expression in human and animal target and nontarget cells can support species selection. This is of special importance when new AAV capsids and regulatory elements like promoters are used as components of the GT. In such cases, small stand‐alone BD studies using qualitative or semiquantitative readouts like bioluminescence imaging may be used to confirm the invitro data before pivotal nonclinical studies with larger numbers of animals are performed[1, 13]. Nonclinical or clinical studies performed with the same AAV capsid and/or pro­moter in combination with another transgene as well as literature data may also help to select a biologically relevant species.

can interfere with the uptake of the functional GT product and reduce exposure of target and nontarget tissues to the GT vector and its expression products. Other contaminations like host cell or helper virus‐derived proteins, miss‐folded pro­teins, aggregates, CpG‐rich DNA impurities might induce additional immune reactions to the administered GT product[16].

A vehicle control group, usually the formulation buffer, should also be included in the nonclinical studies. As the capsid is a component of the AAV GT, which can induce an immune response potentially associated with adverse observations, an empty capsid vehicle control is not warranted.

including GT product properties like vector type/tropism, expression product, ROA as well as disease pathophysiology, existing nonclinical data, and the intended clinical population the core panel may be expanded or even reduced. For example, additional tissues/biofluids may include vitreous fluid, eyes, optic nerve, bone marrow, dorsal root ganglia, cerebrospinal fluid, peripheral nerves, or drain­ing lymph nodes[1]. In cases where systemic exposure is not expected (e.g. sub­retinal administration) collection of a reduced panel of tissues/biofluids might be sufficient. Usually, the vector DNA is measured and, depending on the GT prod­uct, the transgene expression product(s) (RNA, protein) are analyzed in BD sam­ples containing the vector DNA[4, 15].

10, 14 and then weekly, later monthly depending on study duration and shed material in the samples). Duration of the shedding assessment depends on factors like the natural course of infection of the parental virus, persistence of the vector in target and nontarget tissues, and potential immune responses to the GT prod­uct as well as of the duration of the nonclinical pharmacology or toxicology study. The shedding observation period can be shortened for a particular sample type if multiple consecutive negative samples (e.g. >3 consecutive values below LOD) are observed[9, 10].

For replication‐incompetent viral vectors like AAV vectors, vector shedding is sufficiently assessed detecting the vector DNA (incl. the transgene sequence). An additional infectivity assay, as requested for replication‐competent viruses to detect the intact virus, is not required[9, 10].

In a GT development program, a liver targeting AAV2/8 vector was designed to express the human arylsulfatase B (ARSB) under the control of the liver‐specific thyroxine‐binding globulin (TBG) promoter to replace the missing enzyme in patients with Mucopolysaccharidosis Type VI[25]. In this program, a very com­prehensive BD (vector DNA, mRNA, protein) and vector shedding evaluation was performed.

transgene[4]. Although AAV‐based GT products may be associated with a lower risk for third parties as the virus is replication‐deficient and non‐pathogenic, pre­cautions (e.g. contraceptive measures in case the GT product is shed through the seminal fluid) may be warranted[4, 28].

In nonclinical studies, efficacy and safety findings can be correlated to the expo­sure with the vector DNA and/or the transgene product(s). In clinical studies, such a correlation is often not possible as the target tissues/organs as well as most nontarget tissues/organs can only be accessed via biopsies. In most cases, such biopsies are not recommended from ethical perspectives. Therefore, the BD data evaluation in clinical studies often is limited to analysis of blood samples (serum/ plasma, cellular fraction).

dystrophy, biopsies of the muscle were taken to analyze at least the distribution of the transgene protein to the target tissue, but no information on vector copy num­bers in muscle tissue was shared[32].

copies/ml stool. Shedding results in urine also vary depending on the drinking habits of a patient and on the sampling time point, e.g. early in the morning vs. afternoon. Medication intake may also affect urine secretion and thus the shed­ding result. Therefore, varying results in such matrices within or between patients should not be overinterpreted.