Chapter 19
Production of Baculovirus and Stem Cells
for Baculovirus-Mediated Gene Transfer into Human
Mesenchymal Stem Cells
Friederike Eilts, Julie Harnischfeger, Daniel Loewe, Michael W. Wolff,
Denise Salzig, and Peter Czermak
Abstract
The discovery of the genome-editing tool CRISPR-Cas9 is revolutionizing the world of gene therapy and
will extend the gene therapy product pipeline. While applying gene therapy products, the main difficulty is
an efficient and effective transfer of the nucleic acids carrying the relevant information to their target
destination, the nucleus of the cells. Baculoviruses have shown to be very suitable transport vehicles for this
task due to, inter alia, their ability to transduce mammalian/human cells without being pathogenic. This
property allows the usage of baculovirus-transduced cells as cell therapy products, thus, combining the
advantages of gene and cell therapy. To make such pharmaceuticals available for patients, a successful
production and purification is necessary. In this chapter, we describe the generation of a pseudotyped
baculovirus vector, followed by downstream processing using depth and tangential-flow filtration. This
vector is used subsequently to transduce human mesenchymal stem cells. The production of the cells and
the subsequent transduction process are illustrated.
Key words Gene therapy, Cell therapy, Tangential flow filtration (TFF), Baculovirus expression vector
system (BEVS), Pseudotyped baculovirus, Human mesenchymal stem cell (hMSC)
1 Introduction
Gene therapy has evolved as a modern medicinal application [1],
which offers a real cure for currently untreatable cancer types [2] or
hereditary diseases [3]. The genome editing tool CRISPR-Cas9
(clustered regularly interspaced short palindromic repeats
(CRISPR) associated nuclease 9) is revolutionizing gene therapy
and will extend the gene therapy product pipeline. In 2019, 2986
gene therapy products were in clinical studies, a growth by 12%
compared to the preceding year (see Note 1).
The main difficulty in gene therapy is an efficient transfer of the
nucleic acids carrying the relevant information into the nucleus of
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_19, © Springer Science+Business Media, LLC, part of Springer Nature 2021
367
Production of Baculovirus and Stem Cells
for Baculovirus-Mediated Gene Transfer into Human
Mesenchymal Stem Cells
Friederike Eilts, Julie Harnischfeger, Daniel Loewe, Michael W. Wolff,
Denise Salzig, and Peter Czermak
Abstract
The discovery of the genome-editing tool CRISPR-Cas9 is revolutionizing the world of gene therapy and
will extend the gene therapy product pipeline. While applying gene therapy products, the main difficulty is
an efficient and effective transfer of the nucleic acids carrying the relevant information to their target
destination, the nucleus of the cells. Baculoviruses have shown to be very suitable transport vehicles for this
task due to, inter alia, their ability to transduce mammalian/human cells without being pathogenic. This
property allows the usage of baculovirus-transduced cells as cell therapy products, thus, combining the
advantages of gene and cell therapy. To make such pharmaceuticals available for patients, a successful
production and purification is necessary. In this chapter, we describe the generation of a pseudotyped
baculovirus vector, followed by downstream processing using depth and tangential-flow filtration. This
vector is used subsequently to transduce human mesenchymal stem cells. The production of the cells and
the subsequent transduction process are illustrated.
Key words Gene therapy, Cell therapy, Tangential flow filtration (TFF), Baculovirus expression vector
system (BEVS), Pseudotyped baculovirus, Human mesenchymal stem cell (hMSC)
1 Introduction
Gene therapy has evolved as a modern medicinal application [1],
which offers a real cure for currently untreatable cancer types [2] or
hereditary diseases [3]. The genome editing tool CRISPR-Cas9
(clustered regularly interspaced short palindromic repeats
(CRISPR) associated nuclease 9) is revolutionizing gene therapy
and will extend the gene therapy product pipeline. In 2019, 2986
gene therapy products were in clinical studies, a growth by 12%
compared to the preceding year (see Note 1).
The main difficulty in gene therapy is an efficient transfer of the
nucleic acids carrying the relevant information into the nucleus of
Blaine A. Pfeifer and Andrew Hill (eds.), Vaccine Delivery Technology: Methods and Protocols, Methods in Molecular Biology,
vol. 2183, https://doi.org/10.1007/978-1-0716-0795-4_19, © Springer Science+Business Media, LLC, part of Springer Nature 2021
367