Highly Efficient and Non-Integrating Vectors for Generating iPSC

The Challenge and Potential Impact

The proliferative and developmental potential of human stem cells offer virtually unlimited access to the differentiated cells that make up the human body [1]. Stem cell has become one of fastest moving areas biomedical research has ever seen. Tests at all stages ranging from cell differentiation to animal models to human clinical trials have begun within a very short period of time aiming at treating a host of diseases. The excitement generated by the vast potential of stem cells is not only felt by members of the health research community but also has caused great interest and awareness from the general population. Until recently, embryonic stem (ES) cells, derived from the inner mass of blastocysts, have been used for these studies. The use of human embryos in creating human ES cells, however, faces ethical controversies.

As a major advancement in the stem cell field, it has recently been shown that mouse and human differentiated cells may be reprogrammed into stem-like, pluripotent cells by the introduction of defined transcription factors [2-4]. The availability of induced pluripotent stem cells (iPS cells or iPSCs) could provide an ethically acceptable and relatively easy-to-access alternative to human ES cells. Furthermore, it is anticipated that therapies developed with iPSCs could circumvent the problem of tissue rejection following transplantation in patients by creating patient- or even tissue-specific pluripotent stem cells.

Still, great challenges stand between the current methods for generating iPSCs and their therapeutic potential. The use of integrating viral vectors has limited therapeutic potential due to the increased risk of tumor formation. It is therefore important to develop safe, effective and efficient targeting and delivery systems to produce iPSCs.

Because of this importance, multiple methods have been published within the last year that used delivery systems other than the retroviral or lentiviral vectors employed in the original iPSC publications. However, these newly reported methods have not addressed all of the known difficulties facing iPSCs creation. For instance, very low efficiency of transient transfection of selected cDNAs plasmids into primary cells lowers the already abysmal percentage of adult cells that can be reprogrammed with retroviral vectors [5]. Non-integrating adenovirus has a very short cDNA expression time period and thus requires repeated deliveries [6]. The application of episomal expression element such as oriP/EBNA1 helps sustaining longer expression time, but presently it is carried on a plasmid vector and does not improve transfection efficiency. Transposase and the Cre recombinase were used to remove integrated transgenes after the induction is completed, but the integration nevertheless occurred at multiple sites and requires careful and stringent analysis to make sure the reversion is complete; even so, elements of the vector may remain in the iPSCs genome [7, 8].

We wish to take this challenge and use it as an opportunity to develop a synthetic targeting and delivery system that will have the advantages of safe handling, no integration, prolonged and efficient reprogramming gene expression, and high transduction efficiency into broad cell types.

Baculovirus has been used in mammalian cells for many years (e.g. the BacMam system). Engineered baculovirus expression vectors (EBEV) will be exploited as a carrier for reprogramming genes for deriving induced pluripotent cells (iPSCs) from human adult cells. It has been well established that baculovirus can infect mammalian cells with broad tropism yet are very safe for regular laboratory handling.The elements planned for Allele Biotech’s new iPS generating system will be novel in the following aspects:

a) Promoter and mRNA structures for maximum level of cDNA expression in mammalian cells
b) Extended presence in the nucleus for sustained cDNA expression for weeks
c) Cleavable fluorescent protein for cell tracking and sorting
d) Auxiliary packaging constructs for increasing tropism to infect a broad range of human adult cells

Allele Biotech’s Design of Baculovirus for iPSCs

A) Mammalian Expression: In order to express reprogramming cDNAs in human cells, a mammalian promoter cassette will be inserted into the transfer vector to be used with Allele Biotech’s Sapphire Baculovirus genomic DNA. This system, derived from Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), has been provided commercially for 10 years by our team and proved to be one of the best baculovirus systems because of its ease to use and high efficiency. Heterologous promoters have been used by us and others to create baculoviral vectors with dual or triple promoter for protein expression in insect, mammalian, and even bacterial cells such as Novagen’s pTriEx vectors and Allele’s pMBEVS. For the purpose of this proposal, we will remove insect expression cassette altogether and use a CAG promoter for driving expression solely in human cells.

B) Broader Tropism: Baculoviruses has been used for gene delivery to various hepatic and nonhepatic mammalian cells albeit the transduction of certain nonhepatic human cells is about 10-100 fold less effectively than hepatic cells [9]. Incorporating vesicular stomatitis virus G protein (VSVG) in the viral particle surface can greatly increase their effective transduction in a much broader range of mammalian cells [10]. For pseudotyping baculovirus, we will use a coinfection protocol with a recombinant baculovirus expressing VSVG. We expect that incorporating VSVG will ensure that human skin fibroblasts would be infected efficiently for Aim 1; and that different human cells could also be reprogrammed to become iPSCs via the proposed pathway in Aim 3 (see below). Different proteins or peptides might be tried as backup methods for pseudo-typing should VSVG not provide satisfactory results.

C) Fluorescent Marker: We will insert a fluorescent protein (FP) either expressed on a separate cassette or driven by an IRES downstream of reprogramming genes. This feature will facilitate tracking the infected cells, monitoring transgene expression, and enriching infected cells. Allele Biotech is one of the few suppliers/developers of fluorescent proteins. Our exclusive mTFP1 (cyan) and mWasabi (green) proteins are about 3 times brighter than EGFP and true monomers with great photostability and pH insensitivity, which should make them great choices for EBEV.

D) Promoter: The CAG promoter, which has been shown to be a strong promoter in mammalian cells and preferred for BacMam expression, will also be used in the EBEV vectors. We have previously designed pMBVES, a baculovirus vector for glycoprotein expression in mammalian cells that contains such a CAG enhancer/promoter. The CAG composite promoter also encompasses an exon1-intron-exon2 segment that will help mRNA processing and export to cytoplasm for translation. We will clone this fragmentfrom pMBVES into the proposed EBEV vectors.

E) RNA Elements: The 5’ UTR region on the mRNA will be examined and any hairpin structures with ??G < 30 kcal/mol or even <20 kcal/mol but with >65% GC will be disrupted. This step will ensure that maximum production be achieved at the translational level [11]. Post-transcriptional regulatory element (PRE) will be included to further boost gene expression. It has been shown that Woodchuck hepatitis virus (WPRE) increases transgene expression by many folds for various viral vectors, and there has been at least one case for a BacMam vector [12]. WPRE will be cloned from Allele Biotech’s existing HiTiter Lentiviral Vectors and inserted in the 3’ UTR upstream of the SV40 polyA signal.

F) Episomal Expression: Originally derived from Epstein-Barr virus (EBV), phophoprotein nuclear antigen 1 (EBNA1) ensures that oriP-containing DNA replicate once per S-phase during cell circle and is maintained in the nucleus as episomes. Although mostly applied to plasmid vectors, such as in one of the iPSC reports [13], the oriP/EBNA1 system can be incorporated into baculovirus systems for sustained mammalian expression [14]. Aside from episomal maintenance, oriP/EBNA1 could further up-regulate transcription of adjacent genes [14]. For these reasons, we will clone the oriP sequence together with the EBNA1 expression cassette from Allele Biotech’s Phoenix Retrovirus vector pBMN-GFP to EBEV vector.

Bibliography and Reference Cited
1. Thomson, J.A., J. Itskovitz-Eldor, S.S. Shapiro, M.A. Waknitz, J.J. Swiergiel, V.S. Marshall, and J.M. Jones, Embryonic stem cell lines derived from human blastocysts. Science, 1998. 282(5391): p. 1145-7.
2. Takahashi, K. and S. Yamanaka, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006. 126(4): p. 663-76.
3. Takahashi, K., K. Tanabe, M. Ohnuki, M. Narita, T. Ichisaka, K. Tomoda, and S. Yamanaka, Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 2007. 131(5): p. 861-72.
4. Yu, J., M.A. Vodyanik, K. Smuga-Otto, J. Antosiewicz-Bourget, J.L. Frane, S. Tian, J. Nie, G.A. Jonsdottir, V. Ruotti, R. Stewart, Slukvin, II, and J.A. Thomson, Induced pluripotent stem cell lines derived from human somatic cells. Science, 2007. 318(5858): p. 1917-20.
5. Okita, K., M. Nakagawa, H. Hyenjong, T. Ichisaka, and S. Yamanaka, Generation of mouse induced pluripotent stem cells without viral vectors. Science, 2008. 322(5903): p. 949-53.
6. Stadtfeld, M., N. Maherali, D.T. Breault, and K. Hochedlinger, Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse. Cell Stem Cell, 2008. 2(3): p. 230-40.
7. Woltjen, K., I.P. Michael, P. Mohseni, R. Desai, M. Mileikovsky, R. Hamalainen, R. Cowling, W. Wang, P. Liu, M. Gertsenstein, K. Kaji, H.K. Sung, and A. Nagy, piggyBac transposition reprograms fibroblasts to induced pluripotent stem cells. Nature, 2009. 458(7239): p. 766-70.
8. Kaji, K., K. Norrby, A. Paca, M. Mileikovsky, P. Mohseni, and K. Woltjen, Virus-free induction of pluripotency and subsequent excision of reprogramming factors. Nature, 2009. 458(7239): p. 771-5.
9. Stanbridge, L.J., V. Dussupt, and N.J. Maitland, Baculoviruses as Vectors for Gene Therapy against Human Prostate Cancer. J Biomed Biotechnol, 2003. 2003(2): p. 79-91.
10. Tani, H., M. Nishijima, H. Ushijima, T. Miyamura, and Y. Matsuura, Characterization of cell-surface determinants important for baculovirus infection. Virology, 2001. 279(1): p. 343-53.
11. Babendure, J.R., J.L. Babendure, J.H. Ding, and R.Y. Tsien, Control of mammalian translation by mRNA structure near caps. Rna, 2006. 12(5): p. 851-61.
12. Mahonen, A.J., K.J. Airenne, S. Purola, E. Peltomaa, M.U. Kaikkonen, M.S. Riekkinen, T. Heikura, K. Kinnunen, M.M. Roschier, T. Wirth, and S. Yla-Herttuala, Post-transcriptional regulatory element boosts baculovirus-mediated gene expression in vertebrate cells. J Biotechnol, 2007. 131(1): p. 1-8.
13. Yu, J., K. Hu, K. Smuga-Otto, S. Tian, R. Stewart, Slukvin, II, and J.A. Thomson, Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences. Science, 2009.
14. Shan, L., L. Wang, J. Yin, P. Zhong, and J. Zhong, An OriP/EBNA-1-based baculovirus vector with prolonged and enhanced transgene expression. J Gene Med, 2006. 8(12): p. 1400-6.

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Sunday, November 15th, 2009 iPSCs and other stem cells

1 Comment to Highly Efficient and Non-Integrating Vectors for Generating iPSC

February 5, 2010

In a recent Nature Method review article on iPS cells, it was stated that “the most intense interest is in ‘vector free’ systems, in which no integration ever takes place…” includes “adenovirus-based approaches or the BacMam system” BacMam refers to using baculovirus on mammalian host cells as we proposed above.

Link: http://www.nature.com/nmeth/journal/v7/n1/full/nmeth0110-81.html

Allele Biotech is listed on top of the iPSC suppliers guide in Table 1.

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