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ADDRESSING THE CHALLENGE OF ENCAPSIDATED HOST CELL DNA
Adeno-associated virus (AAV)-based vectors have emerged as the preferred delivery tool for in vivo gene therapies. One concern with the use of such vectors are possible process- and product-related impurities. An important example of the latter is encapsidated host cell DNA (hcDNA). To overcome this challenge, CEVEC has developed an approach that drastically reduces the amount of this contaminant in recombinant AAV (rAAV) preparations. The process is universally applicable for transient and stable AAV production systems. In particular in combination with CEVEC’s inducible stable ELEVECTA® producer cells1, this provides a valuable platform for large scale production of AAV-based vectors and is another important step towards making AAV-based gene therapies even safer.
USE OF AAV FOR IN VIVO GENE THERAPY
The rapid increase in the use of AAV-based vectors for in vivo gene therapies in recent years is due to a number of favorable properties of this vector type2. These include, for example, the advantageous safety profile, the fact that AAV is not pathogenic, the persistence of the viral genome, the concomitant stable gene expression, as well as the ability to transduce dividing and non-dividing cells. Furthermore, the tropism, i.e. the host cell specificity, can be controlled in a desired way by selecting suitable naturally occurring or engineered capsids. Accordingly, the number of clinical trials conducted with rAAV has been steadily increasing. The crucial importance of these vectors is also reflected by the fact that, with Glybera, Luxturna and Zolgensma, most in vivo gene therapies that have been approved in the Western world rely on AAV-based vectors and it is to be expected that numerous similar therapies will be approved in the near future2.
PRODUCTION OF AAV-BASED VECTORS
AAV-based vectors are normally produced in mammalian (typically human) or insect cells3,4. Commonly used human cell lines are HEK-293, HeLa or CEVEC's proprietary CAP cells. In these cases, the genetic elements necessary for AAV production are introduced into the host cell by transient transfection of plasmid DNA, transduction with a helper virus or stable integration into the host genome. These genetic elements include the AAV transfer vector with the inverted terminal repeats (ITRs)-flanked gene of interest (GOI), the sequences coding for the required AAV proteins and the adenoviruses or herpesviruses-derived helper-functions that are needed for AAV genome replication. Alternatively, insect cells can be used for rAAV production, in which case the necessary genetic elements are transduced into Spodoptera frugiperda cells via recombinant baculoviruses.
ENCAPSIDATED hcDNA IN rAAV PREPARATIONS
A remaining challenge for the use of rAAV in gene therapy is the identification, characterization and control of impurities in vector preparations. While impurities are a concern for every drug substance, they constitute a particular two-part problem in AAV preparations, where contaminants might get carried over outside and inside of the viral capsid. Unpackaged host cell components like DNA, RNA, proteins and lipids are termed process-related impurities and they can be effectively removed by conventional downstream process purifications5. Product-related impurities on the other hand, involve host cell components that are incorporated in the viral capsid and are therefore intrinsically resistant to classic purification methods. Among these encapsidated impurities, hcDNA represents the most problematic stowaway. Encapsidated hcDNA might arise when fragments of host cell genomic DNA, rather than the virus genome, are unintentionally enclosed into virus particles (Figure 1).
Figure 1: Emergence of hcDNA-containing AAV particles exemplified by production in a mammalian cell. The required genetic elements (left) are introduced into a mammalian producer cell (middle), for example by transient transfection of plasmid DNA, transduction with a helper virus or stable insertion into the host genome. While most genome-containing AAV particles will contain the AAV transfer vector with the GOI, hcDNA (red) may be encapsidated in a small proportion (right). Note that some particles may also exist as empty capsids, which may be removed during the purification process (not shown).
The underlying mechanisms are poorly understood, but it appears that the great excess of hcDNA relative to the AAV genome template in the producer cells during the manufacturing process contributes to the incorrect packaging of hcDNA fragments.It has been reported that in purified preparations from mammalian producer cells 1-3% of all genome-containing AAV particles comprise hcDNA fragments due to such events6.
Safety concerns associated with encapsidated hcDNA relate to potential genotoxicity on the one hand and immunotoxicity on the other5. An example of the former would be the packaging of an oncogene into AAV particles, whereas the latter might result from adverse immune effects due to the presence of sequences that code for immunogenic proteins. The relative risk for the one or the other might depend on the origin of the AAV producer cell, i.e. mammalian vs. insect cells. In any case these concerns led to FDA recommendations according to which the level of residual cell-substrate DNA should be below 10 ng per dose, with a median DNA size of 200 bp or lower5. Therefore, compliance with these limits must be taken into account when producing AAV-based vectors.
MINIMIZING THE AMOUNT OF ENCAPSIDATED hcDNA
In order to address this challenge, CEVEC has developed a breakthrough approach that reduces the abundance of hcDNA-containing particles already during rAAV production. As shown in Figure 2, this optimized process reduces the amount of encapsidated hcDNA impurities in both, transient, as well as stable platforms, such as CEVEC’s ELEVECTA® system.
Figure 2: Improved production process reducing hcDNA-containing AAV particles across various production platforms. Applying CEVEC’s proprietary process reduces hcDNA content in AAV particles by about 2 log units in CAP cells, independently from whether the manufacturing platform is based on Cevec’s stable ELEVECTA® system (columns 1 and 2) or a production using transient triple transfection of plasmids (column 3 and 4).
The process is non-toxic to AAV producer cells, does not require elaborate instrumentation or hands-on time and is compatible with high viral titers. In combination with CEVEC’s stable ELEVECTA® producer cells, the new optimized production process meets the steadily growing demand for high volumes of rAAV preparations and scalability, while simultaneously addressing the continuously increasing safety requirements for industrial AAV production.