Optimization of adeno-associated virus (AAV) gene delivery into human bone marrow stem cells (hBMSCs)

Abstract

Background Efficiently delivering nucleic acid into mammalian cells is essential to overexpress genes for assessing gene functions. Human bone marrow stem cells (hBMSCs) are the most studied tissue-derived stem cells. Adeno-associated viruses (AAVs) have been used to deliver DNA into hBMSCs for various purposes. Current literature reported that transduction efficiencies of up to 65% could be achieved by AAV gene delivery into hBMSCs. Further improvement of efficiency is needed and possible. This study tested a selection of AAV serotypes for high-efficient DNA delivery into hBMSCs. Methods hBMSCs from different donors were infected with different serotypes of AAVs containing the enhanced green fluorescence protein ( eGFP ) reporter gene driven by the CMV promoter. Green fluorescence was monitored in the infected cells at five-day intervals. Cells were collected at designated time points after the infection for reverse-transcription polymerase chain reaction (RT-PCR) and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) to assess eGFP mRNA transcription. Results The results indicated that the order of transduction efficiency of the AAV serotypes was AAV2 > AAV2.7m8 > AAV6 > AAV6.2 > AAV1 > AAV-DJ. AAV2 could achieve almost 100% transduction at the multiplicity of infection (MOI) greater than 100K. Over 90% of cells could be transduced at 20K to 50K MOI. About 80% transduction was seen at MOIs of 10K and 15K. RT-PCR analysis showed that eGFP mRNA could be detected from day 5 to day 30 post-AAV infection. The differences in the observed transduction efficiencies of the hBMSCs from different patients indicate donor-to-donor variability, and increased eGFP mRNA was generally seen after day 15 post-AAV2 infection. Maximal eGFP transcription was detected on day 30 post-infection. Conclusions We conclude that AAV2 and AAV2.7m8 at an MOI of 100K or greater can efficiently deliver transgene into hBMSCs with up to near 100% transduction efficiency for sustained expression over one month. However, donor-to-donor variation exists in transduction efficiency and transgene expression, especially at MOIs less than 100K.

Genetic and Functional Analyses of Patients with Marked Hypo-High-Density Lipoprotein Cholesterolemia

“…genomic solution at a concentration of 1.0 x 10 12 GC/ mL was mixed with 140 µL of the medium and applied. After overnight incubation, the medium was changed every 2 days and evaluated on day 7 [21][22][23][24] .…”

Adenovirus-Based Gene Therapy for Bone Regeneration: A Comparative Analysis of In Vivo and Ex Vivo BMP2 Gene Delivery

“…For example, Blum et al demonstrated that MSCs genetically modified with Ad-BMP2 have a higher osteogenic capacity then cells infected with lentiviral constructs or lipoplexes [ 28 ]. At the same time, adeno-associated viruses have significant limitations for gene therapy of bone regeneration due to their low ability to transduce osteogenic precursors such as bone marrow-derived MSCs [ 29 ] and the high variability in infection efficiency from donor-to-donor [ 30 ].…”

Designing molecules: directing stem cell differentiation

“…AAV has many serotypes and variants and different AAV serotypes exhibit significant differences in the transduction efficiencies and cell and tissue tropism ( Yao et al, 2023 ). Selection of the appropriate AAV serotype is one of the first steps in designing AAV gene delivery ( Wiley et al, 2018 ).…”

“…Although this mutant showed a slightly higher affinity for heparin compared to AAV2, it is observed similar levels of binding of AAV1.9 and AAV2 to hESCs ( Asuri et al, 2012 ). In another study, AAV2 is highly efficient in delivering genes into human bone marrow stem cells (hBMSCs) ( Yao et al, 2023 ). High transduction efficiencies (>80%) can be achieved by adding virus particles to cell cultures at MOI OF 10,000.…”

“…When hBMSCs are infected with higher MOI (>100,000) of AAV2 or its mutant AAV2.7m8, the transduction efficiency can reach nearly 100%. However, the result suggest that genetic background of cell donor may influence AAV transduction efficiency, especially at low MOI (Yao et al, 2023). can be identified and isolated from supernatants and body fluids.…”