Challenges in lentiviral Cas9 packaging

Having established the functionality of the newly generated SAM split-dCas9-VPR system by lipofection and RT-qPCR the next step was lentiviral production for reprogramming experiments. However, in several attempts lentiviral titers were low with only 1 x 10⁵ - 1 x 10⁶ total infectious particles per harvest whereas former harvests e.g. for ALN lentiviruses were in the range of 1 x 10¹⁰ - 1 x 10¹¹ viral particles. With such a low titer a complete harvest (1.5 x 10⁵ viral particles) would be required for a single well of a 24-well plate in order to transduce cells at a multiplicity of infection of three (MOI 3). Titers from colleagues also working with lentiviral Ef1a-Cas9 vectors were comparably low suggesting possible adverse effects of the dCas9 coding sequences or gRNA secondary structures for lentiviral packaging (personal communication). This was surprising, as similar designs have been published comprising e.g. an Ef1a promoter and dCas9-VP64 [91]. However, in this publication selection markers were used to obtain a stable cell line where low numbers of transduced cells are not the limiting factor. Unfortunately, such a selection process is not suitable for reprogramming of primary cells (unpublished experimental data of collaborating group).

One alternative to viral delivery of Cas9 would be lipofection which was utilized for the conversion of embryonic stem cells to extraembryonic lineages [106]. However, the efficiency of gene delivery in astrocytes was found to be << 1% by lipofection (approximately 70 ± 20 total cells 14 days after seeding 5 x 10⁴ cells, data not shown) compared to approximately 30 – 40% transduced cells by lentiviruses. This underlines the need for an efficient lentiviral delivery system.

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Therefore, one idea was to invert the cassette including the gRNA sequence, dCas9 components and activator complexes in order to prevent unintentional translation from the viral RNA during packaging or possible inhibitory effects due to secondary structures of the gRNA. However, a first RT-qPCR analysis suggested a strong decrease in Ascl1 induction from 4.7 ± 0.1 x 10³ to 0.30 ± 0.02 x 10³-fold upon inversion of the cassette as depicted Figure 22A. This system was therefore not used for further tests. Another possible solution was the exchange of the Ef1a promoter as Black et al., [107] report in a current article for the first time reprogramming of fibroblasts to neurons using dCas9 expressed from lentiviruses by a human Ubiquitin C (hUBC) promoter. This was surprising as hUBC is a comparably weak promoter [120] and it was expected that high levels of expression would be beneficial for successful reprogramming (see Figure 8B and C). Nevertheless, sufficient lentiviral packaging must have been accomplished by Black et al., [107].

Therefore, lentiviral production was repeated with a hUBC promoter and additionally a Tet-O promoter as an alternative known for its high levels of expression [120]. Interestingly, the replacement of the Ef1a promoter increased lentiviral titers to 3 x 10⁸ (hUBC-Cas9) and 1 x 10⁹ (Tet-O-Cas9) total viral particles per harvest. As expected, Tet-O-Cas9 was expressed at much higher levels than hUBC-Cas9 which was close to the detection limit of the immunocytochemistry analysis depicted in Figure 22C.

The Tet-O construct was chosen for further experiments due to its high level of expression and the possibility to influence timing and activity by adjusting the doxycycline concentration (see chapter 5.1.5). A RT-qPCR screen shown in Figure 22B comparing the SAM split-dCas9-VPR system using Ef1a or Tet-O promoters confirmed the functionality of the new Tet-O constructs. Western blot analysis in Figure 22D using a Cas9 antibody revealed successful generation of N-dCas9 at 80 kDa from newly generated viruses with comparable band patterns and intensities to transfected cells. A fraction of total N-dCas9 was found as a fusion protein with SAM at 130 kDa due to inefficient ribosome skipping at the P2A site separating SAM and N-dCas9. Upon addition of C-dCas9-VPR viruses a shift in size was observed indicating successful assembly of N- and C-dCas9 parts at 218 kDa.

These findings were confirmed by visualization of C-dCas9-VPR via a MycTag in a second western blot in Figure 22E. The newly generated SAM split-dCas9-VPR viruses were therefore successfully packaged and dCas9-VPR was able to assemble itself after transduction.

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Figure 22: Replacement of Ef1a by Tet-O or hUBC promoters enables lentiviral packaging of dCas9 (A) RT-qPCR analysis of Ascl1 induction in Neuro 2a cells 48 h after transfection. Inversion of the expression cassette of split-dCas9-VPR (hU6-mA1-Ef1a-SAM-N-dCas9 + hU6-mA2-Ef1a-C-dCas9-VPR) resulted in a strong decrease in Ascl1 expression. In this case, expression cassettes including promoters and gRNAs were inverted relative to the lentiviral backbone (see Figure 20C for comparison). (B) RT-qPCR of Ascl1 induction comparing split-dCas9-VPR system with Ef1a or Tet-O-promoters revealed similar levels of activation for the two promoters. (C) Immunocytochemistry anlysis of Cas9 in HEK293 cells 48 h after transduction by 0.5 µl lentiviruses encoding hUBC-Cas9 or Tet-O-Cas9. While the percentage of Cas9⁺/DAPI⁺ cells was comparable the expression level in hUBC-Cas9 transduced cells was much lower as suggested by the different intensities of the Cas9 signal. (D) Western blot analysis using an α-Cas9 antibody detecting N-terminal Cas9 only. Viruses were functional as assessed by comparable bands of transfected and transduced cells. The P2A splicing between SAM and N-dCas9 was not complete as expected. However, with the majority of N-dCas9 correctly cleaved. Upon adding dCas9-VPR N- and C-parts assembled to dCas9-VPR at 218 kDa. (E) Western blot analysis using an α-MycTag antibody for the detection of C-dCas9-VPR. The C-dCas9-VPR virus was functional with protein bands similar to transfected cells showing successful assembly of dCas9-VPR upon addition of N-dCas9. Abbreviations:

C-dCas9: C-terminal dCas9 residues 574-1368, Ef1a: Eukaryotic translation initiation factor 1A promoter, N-dCas9: N-terminal dCas9-residues 1-573, SAM: MS2-P65-HSF1 fusion protein, hUBC: human ubiquitin C promoter, VPR: VP64-P65-RTA fusion protein. Scale bars: 50 µm. Data was derived from one experiment.

Error bars represent mean ± SEM.

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5.2.5 Direct reprogramming of astrocytes to neurons utilizing SAM and VPR