Transduction Enhancers Enable Efficient Human Adenovirus Type 5-Mediated Gene Transfer into Human Multipotent Mesenchymal Stromal Cells

Abstract

Human multipotent mesenchymal stromal cells (hMSCs) are currently developed as cell therapeutics for different applications, including regenerative medicine, immune modulation, and cancer treatment. The biological properties of hMSCs can be further modulated by genetic engineering. Viral vectors based on human adenovirus type 5 (HAdV-5) belong to the most frequently used vector types for genetic modification of human cells in vitro and in vivo. However, due to a lack of the primary attachment receptor coxsackievirus and adenovirus receptor (CAR) in hMSCs, HAdV-5 vectors are currently not suitable for transduction of this cell type without capsid modification. Here we present several transduction enhancers that strongly enhance HAdV-5-mediated gene transfer into both bone marrow- and adipose tissue-derived hMSCs. Polybrene, poly-l-lysine, human lactoferrin, human blood coagulation factor X, spermine, and spermidine enabled high eGFP expression levels in hMSCs. Importantly, hMSCs treated with enhancers were not affected in their migration behavior, which is a key requisite for many therapeutic applications. Exemplary, strongly increased expression of tumor necrosis factor (TNF)-stimulated gene 6 (TSG-6) (a secreted model therapeutic protein) was achieved by enhancer-facilitated HAdV-5 transduction. Thus, enhancer-mediated HAdV-5 vector transduction is a valuable method for the engineering of hMSCs, which can be further exploited for the development of innovative hMSC therapeutics.

Hexon modification of human adenovirus type 5 vectors enables efficient transduction of human multipotent mesenchymal stromal cells

“… 2 , 30 However, due to the lack of CAR expression, hMSCs are very difficult to transduce with HAdV-5 vectors. As we previously observed that positively charged molecules significantly enhanced HAdV-5-mediated gene transfer into hMSCs, 27 we analyzed transduction of bone marrow-derived (BM-) and adipose tissue-derived (A-) hMSCs with the charge-modified vectors HAdV-5-ΔHVR1, HAdV-5-HexPos2, and HAdV-5-HexPos3. We observed a striking 500-fold increase in mean fluorescence intensity (MFI) of BM-hMSCs after transduction with HAdV-5-HexPos3 compared with HAdV-5 ( Figure 2 A).…”

“… 59 , 60 However, ex vivo transduction of hMSCs with HAdV-5-based vectors is very inefficient due to a lack of CAR expression on this cell type. 13 , 27 We recently identified several transduction enhancers that can be used to enhance HAdV-5-mediated gene transfer into hMSCs. 27 However, while transduction enhancers are a readily available adjuvant for in vitro transduction experiments, prior to clinical use, a potential impact on the biology and functionality would need to be considered.…”

“… 13 , 27 We recently identified several transduction enhancers that can be used to enhance HAdV-5-mediated gene transfer into hMSCs. 27 However, while transduction enhancers are a readily available adjuvant for in vitro transduction experiments, prior to clinical use, a potential impact on the biology and functionality would need to be considered. In addition, using transduction enhancers as part of a production process under good manufacturing process conditions would increase process complexity, potentially negatively affect scalability and reproducibility, and significantly increase overall costs.…”

“…As MSCs lack expression of the coxsackie and adenovirus receptor (CAR)—the primary attachment receptor of HAdV-5 particles in vitro —transduction of hMSCs with HAdV-5 vectors has been very inefficient. 13 , 25 , 27 As we have shown recently, one way to address this bottleneck is by using positively charged transduction enhancers to enable uptake of the negatively charged HAdV-5 vector particles into human (h)MSCs. 27 However, as transduction-enhancing agents might affect the biology of hMSCs and, importantly, their use would increase the complexity of the manufacturing process, application of genetically capsid-modified adenovirus vectors efficiently transducing hMSC would clearly be preferable.…”

“… 13 , 25 , 27 As we have shown recently, one way to address this bottleneck is by using positively charged transduction enhancers to enable uptake of the negatively charged HAdV-5 vector particles into human (h)MSCs. 27 However, as transduction-enhancing agents might affect the biology of hMSCs and, importantly, their use would increase the complexity of the manufacturing process, application of genetically capsid-modified adenovirus vectors efficiently transducing hMSC would clearly be preferable. We rationally designed HAdV-5 mutant vectors by replacing surface-exposed negatively charged amino acids (aa) with positively charged lysines.…”