<figure><img src="image_1.png" /></figure>

HAL
open science

# Canine helper-dependent vectors production: implications of Cre activity and co-infection on adenovirus propagation

P. Fernandes, A. I. Almeida, E. J. Kremer, P. M. Alves, A. S. Coroadinha

► To cite this version:

P. Fernandes, A. I. Almeida, E. J. Kremer, P. M. Alves, A. S. Coroadinha. Canine helper-dependent vectors production: implications of Cre activity and co-infection on adenovirus propagation. Scientific Reports, 2015, 5, pp.9135. 10.1038/srep09135 . hal-02187392

HAL Id: hal-02187392

https://hal.science/hal-02187392

Submitted on 27 May 2021

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_2.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
<figure><img src="image_3.png" /></figure>

OPEN

SUBJECT AREAS:
GENE THERAPY
CELL BIOLOGY
ADENOVIRUS
MOLECULAR BIOLOGY

Received
27 October 2014

Accepted
20 February 2015

Published
16 March 2015

Correspondence and
requests for materials
should be addressed to
A.S.C. (avalente@itqb.unl.pt)

# Canine helper-dependent vectors production: implications of Cre activity and co-infection on adenovirus propagation

Paulo Fernandes¹,², Ana I. Almeida¹, Eric J. Kremer³, Paula M. Alves¹,² & Ana S. Coroadinha¹,²

¹iBET, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal, ²Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal, ³Institut de Génétique Moléculaire de Montpellier, CNRS – Universities of Montpellier I and II, Montpellier, France.

The importance of Cre recombinase to minimize helper vector (HV) contamination during helper-dependent adenovirus vectors (HDVs) production is well documented. However, Cre recombinase, by inducing DNA double-strand breaks (DSBs), can cause a reduced proliferation and genotoxic effects in cultured cells. In this work, Cre-expressing cell stability, co-infection and their relation to adenovirus amplification/HV contamination were evaluated to develop a production protocol for HD canine adenovirus type 2 (CAV-2) vectors. Long-term Cre expression reduced the capacity of MDCK-E1-Cre cells to produce CAV-2 by 7-fold, although cell growth was maintained. High HDV/HV MOI ratio (5 : 0.1) led to low HV contamination without compromising HDV yields. Indeed, such MOI ratio was sufficient to reduce HV levels, as these were similar either in MDCK-E1 or MDCK-E1-Cre cells. This raises the possibility of producing HDVs without Cre-expressing cells, which would circumvent the negative effects that this recombinase holds to the production system. Here, we show how Cre and MOI ratio impact adenovirus vectors yields and infectivity, providing key-information to design an improved manufacturing of HDV. Potential mechanisms to explain how Cre is specifically impacting cell productivity without critically compromising its growth are presented.

Adenoviruses have received considerable attention as vectors for gene therapy and their genome has been progressively modified to improve safety and efficacy in therapeutic applications. From vectors with the deletion of the E1 region to helper-dependent vectors (HDVs) with the deletion of all viral genes, an enhanced capacity for a gene therapeutic insertion from ~7 kb to ~36 kb has been achieved¹. Most replication-defective adenovirus vectors require for manufacturing and replication a cell line that expresses the adenoviral E1 functions in *trans*. However, since HDVs retain only the inverted terminal repeats and the packaging signal, early and late viral functions need to be provided in a synchronized fashion. Standard productions of HDVs are based on co-infection of HDV and a helper vector (HV), which provides all viral proteins in *trans*. During production, both HDV and HV genomes replicate inside the cells, but the encapsidation of the HV genome is minimized, by flanking its packaging signal (ψ) with recognition sequences for a recombinase constitutively expressed by the cells, such as Cre². Therefore, the E1 transcomplementing cell line for HDV production must also express the recombinase²⁻⁴. Cre/loxP system was the first system described to efficiently reduce HV contaminant, and is still the most common system used for HDV propagation.

The importance of high levels of Cre to avoid HV propagation is unquestionable⁵. In fact, considering that the remaining HV contaminant was due to limited Cre activity levels that permitted HV to escape packaging signal excision⁵, major advances were made to increase the levels of recombinase during HDV production⁶. On the other hand, the effect of Cre expression on producer cell homeostasis has been undervalued. Cre expression can result in a markedly reduced proliferation and genotoxic effects in cultured cells⁷. The aberrant activity on multiple pseudo loxP sites presented in the mammalian genome⁸ induces DNA double-strand breaks (DSBs) or nicks that are converted to DSBs during DNA replication, leading to arrest of cells in G₂/M phase because of intolerable DNA damage⁷. Furthermore, although sustained low-levels might be supported by cells without significant toxicity, they cause a slow, cumulative increase in recombination and chromosome abnormalities. Therefore, the stability
of cells constitutively expressing Cre recombinase is likely to be impaired. While these effects are not substantiated in literature in the scope of adenovirus production, they are considerably described for several cell lines$^{7,9-11}$, including HEK 293 cells$^9$, traditionally used to propagate adenoviruses. From a bioprocess point-of-view, failure of producer cell line robustness to maintain performance compromises the viral preparations quality, process yields, reproducibility, economics and the regulatory approval.

Previously, we established MDCK-derived cell lines for the pro-
duction of canine adenovirus type 2 (CAV-2) vectors¹². In this work,
we studied the impact of Cre recombinase in the scope of an optimal
production process for HD CAV-2 with MDCK-derived cells in
serum-free medium, emphasizing two aspects: cell line stability
and infection conditions. Canine adenovirus type 2 (CAV-2) vectors
were selected because of their attractive features to bypass the clinical
disadvantages of using human adenoviruses while keeping other
advantages¹³,¹⁴, to address fundamental neurobiological ques-
tions¹⁵⁻¹⁹ and to develop potential treatment of neurodegenerative
and ocular disorders²⁰⁻²². Moreover, MDCK cells are already
approved by the regulatory authorities for the manufacture of vac-
cines and thus represent a suitable cell substrate that might facilitate
the approval of clinical grade CAV-2 vectors production²³. MDCK-
E1-Cre cell line stability was addressed by evaluating growth, Cre
activity levels and virus production in cells with increasing culture
passages. Different HDV/HV MOI ratios were assayed to determine
the best infection conditions to amplify HD CAV-2 and to under-
stand the relation between Cre expression, infection conditions and
HV propagation. Here, we show how Cre expression impacts cell
performance and adenovirus propagation and how infection condi-
tions significantly contribute to reduce HV contamination during
HDV production even in the absence of Cre-recombinase.

Results

MDCK-E1-Cre cells, once adapted to grow in Optipro SFM, were
maintained in culture for 20 more passages (P) in duplicates, with
and without antibiotic pressure, and frozen at P0, P10 and P20 (see
material and methods section). MDCK-E1 cells, subjected to same
procedure, were used as a control for CAV-2 vector production. The

experiments to determine the best infection conditions for HD
CAVGFP (HDV) production were performed with cells at P0.

**Cell line evaluation at different culture passages.** *Growth characteristics of MDCK-E1-Cre.* Growth curves of MDCK-E1-Cre cells obtained in the different cell culture passages were performed in 25 cm² t-flasks. Concentration and viability were determined every day (Figure 1).

The results show that cell growth was similar in all culture pas-
sages, regardless of antibiotic pressure. On average, MDCK-E1-Cre
cells showed a specific growth rate of 1.00 ± 0.07 day⁻¹ (Figure 1A)
and a minimum cell viability of 63 ± 3% obtained during stationary
phase (Figure 1B). These results indicate that increasing passages and
selective pressure had no impact on MDCK-E1-Cre cell growth.

*Cre expression and excision efficiency of MDCK-E1-Cre.* To evaluate the impact of increasing passages in Cre expression, two approaches were used. The first estimates Cre activity through the level of luciferase activated by the excision of a stop sequence flanked by loxP sites (view materials and methods section) (Figure 2). In the second approach, Cre activity was estimated by evaluating the reduction in HV JBΔ5 propagation (due to excision of packaging genome) in MDCK-E1-Cre cells (Table 1). The results obtained from both methods were similar. A slight decrease in Cre activity was obtained in cells at P20 when maintaining the antibiotic pressure (Figure 2), nevertheless this reduction was not statistically significant nor significantly altered the HV excision efficiency (Table 1). Without the selective pressure, a further 2.5 ± 0.5 fold decrease in Cre activity was observed from P10 to P20 (Figure 2), which contributed to approximately 3-fold reduction in the capacity for HV genomes excision (Table 1).

CAV-2 production of MDCK-E1-Cre. To evaluate the impact of increasing subculture passages on the capacity to produce viral vectors, production assays with CAVGFP (ΔE1) were performed. MDCK-E1 cells maintained in parallel under same passages number were added to the experiments and used as controls for ΔE1 production.

In MDCK-E1 cells, an increase of 2.1 ± 0.3 fold in productivity
was observed with increasing cell passages and regardless of selective

<figure><img src="image_4.png" /></figure>

**Figure 1 |** Effect of cell culture passage and antibiotic pressure on MDCK-E1-Cre cell growth (A) and viability (B). Dashed line represents the average of data points and error bars the corresponding standard-deviation. MDCK-E1-Cre cell growth was similar to MDCK-E1 cells<sup>23</sup>; for a matter of simplicity data points for MDCK-E1 cells are not shown. w/: with antibiotic pressure; w/o: without antibiotic pressure.
<figure><img src="image_5.png" /><figcaption>**Figure 2 | Effect of cell culture passage and antibiotic pressure on Cre-activity of MDCK-E1-Cre cells.** Light units were normalized to cell concentration and background obtained with MDCK-E1 cells. Values are shown as average ± standard-deviation (n = 3). w/AB: with antibiotic pressure; w/o AB: without antibiotic pressure. * *p* = 0.01, given by a single factor Anova analysis against the corresponding P0 values.</figcaption></figure>

pressure (Figure 3). Such feature has been reported for these cells¹². On the other hand, MDCK-E1-Cre cells showed a decrease in the production of ΔE1. A slight reduction of 1.6 ± 0.5 fold was observed in cells without selective pressure at P10, in which productivity was then maintained up to P20. On the other hand, MDCK-E1-Cre under selective pressure showed a consecutive reduction in ΔE1 production capacity with increasing passages, achieving a 7.1 ± 1.7 fold-decrease at P20. These observations indicate that production capacity of cells was hampered by maintaining Cre activity through selective pressure.

**Establishing the best conditions for HD CAV-2 production.** To exclude any cell passage effect, the following experiments were performed in MDCK-E1-Cre and MDCK-E1 cells at P0.

**HDV/HV MOI ratio.** To establish optimal conditions to produce HDV (maximum productivity and lowest HV contamination), production assays under different MOI ratios (5 to 1, 5 to 0.5, and 5 to 0.1 IP) were performed. MOI ratio of 5 to 5 was initially tested, but critically compromised cell viability and production of either HDV or HV (data not shown).

Similar HDV specific productivities of approximately 300 IP/cell were obtained using the different MOI ratios tested (Figure 4A). Even with a MOI as low as 0.1, HDV productivity was still maintained.

When using MDCK-E1-Cre cells, HV contamination levels were similar in the three co-infections (2–3%) (Figure 4B).

To further understand how Cre recombinase impacts HV propagation in the different MOI ratios, the assays were also performed in MDCK-E1 cells (Figure 4A, B). The results show a similar HDV production in both cells (Figure 4A), however a clear proportion between HV MOI and its contaminant levels was observed (Figure 4B). While at MOI 1 of HV a high contamination was observed (16.3%), a decrease in HV contaminant to 2.0% was possible when decreasing MOI of this vector. Moreover, contaminant levels of HV with MDCK-E1 cells in 5HDV: 0.1HV co-infections were similar to those obtained with MDCK-E1-Cre cells. These results indicate that co-infection with the lowest HV MOI tested herein, i.e. 0.1 of HV, was sufficient to minimize contamination during HDV production.

**The impact of MOI ratio in co-infections.** Given that HDV production requires co-infection with HV, the impact of co-infection conditions on adenovirus propagation and HV contaminant levels were further analyzed using other CAV-2 vector. Infections assays under different MOI ratios were reproduced by replacing HDV for ΔE1 CAV-2 vectors. Similarly to HDV assays, cells were co-infected with ΔE1 (MOI 5) and HV at MOI 5, MOI 1 or MOI 0.5 using MDCK-E1 and MDCK-E1-Cre cell-lines.

The production of ΔE1 vector increased when HV MOI was decreased for both cell lines (Figure 4C). However, MDCK-E1-Cre cells showed lower capacity to produce CAVGFP than MDCK-E1 cells, especially under high virus input. Similarly to the assays with HDV, the proportion of HV was minimal in MDCK-E1-Cre cells, and highly dependent on the MOI when MDCK-E1 cells were used (Figure 4D). However, in each infection condition assayed, a constant fold-reduction (5- to 6-fold) in HV contamination levels was observed when comparing MDCK-E1 to MDCK-E1-Cre cells. These results corroborate that the MOI ratio used to infect cells determines the proportion of each vector in the final production pool, i.e. which vector and its propagation is favored.

**The role of Cre recombinase and MOI on the propagation of HV.** To gather a better understanding of the factor behind a low HV contamination in HDV production, the role of Cre recombinase and MOI were evaluated from an HV propagation point-of-view. To understand the relation between Cre recombinase and HV propagation under different MOIs, infections with HV using MOI 5, 1 and 0.5 were assayed in MDCK-E1-Cre and compared to those obtained with MDCK-E1 cells (Figure 5). A decrease in HV production was observed in MDCK-E1-Cre cells in comparison to MDCK-E1 cells due to excision of the packaging signal by Cre activity. Cell specific productivity was proportional to the MOI used in both cell lines. Furthermore, the differences in IP/cell of MDCK-E1-Cre versus MDCK-E1 cells in the different MOIs tested were shown to be similar, following on average a fold-reduction of 145 ± 23. These observations indicate that the number of HVs escaping from excision was proportional to the MOI used.

<table><caption>**Table 1 | Effect of cell culture passage and antibiotic pressure on excision efficiency of HV genomes in MDCK-E1-Cre cells**</caption><thead><tr><th rowspan="2">Passages</th><th colspan="2">Excised/unpackaged HV genomes (%)</th></tr><tr><th>w/AB</th><th>w/o AB</th></tr></thead><tbody><tr><td>P0</td><td></td><td>98.9 ± 0.6</td></tr><tr><td>P10</td><td>98.4 ± 1.5</td><td>97.7 ± 2.3</td></tr><tr><td>P20</td><td>92.4 ± 7.2</td><td>28.5 ± 2.3</td></tr></tbody></table>

Values are shown as average ± standard-deviation (n = 2). Since MDCK-E1 and MDCK-E1-Cre cells have similar productivities at P0 (Figure 3), the HV amplification reduction obtained in MDCK-E1-Cre cells corresponds to Cre-mediated excision of HV genomes avoiding their packaging. The percentage of excised HV genomes was estimated assuming that the differences of HV amplification titers between (a) MDCK-E1 and (b) MDCK-E1-Cre cells corresponds to excised/unpackaged HV genomes as follows: [(a - b)/a]. Value (b) was also normalized to the fold-reduction of MDCK-E1-Cre cells productivity observed during passages (Figure 3). w/AB: with antibiotic pressure; w/o AB: without antibiotic pressure.
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 3** | Effect of cell culture passage and antibiotic pressure on MDCK-E1-Cre cells specific productivity. As a control, MDCK-E1 cells under same conditions were added to the experiments. Values are shown as average ± standard-deviation (n = 3). w/: with antibiotic pressure; w/o: without antibiotic pressure. \* *p* < 0.05, \*\* *p* < 0.005, given by a single factor Anova analysis against the corresponding P0 values.</figcaption></figure>

*The relation between viral particles infectivity, Cre and packaging.* Physical (genome-containing) particles of HDV, ΔE1 and HV were quantified and normalized to the infectious titer to analyze the infec- tivity in the different conditions assayed.

The results show a higher PP:IP ratio for HDVs than for ΔE1 (Figure 6), as previously described²⁴. When using MDCK-E1 as pro- ducer cells, the PP:IP ratios of HDV and ΔE1 were similar along the different co-infections (Figure 6A, B). However, HDV and ΔE1 PP:IP ratios obtained with MDCK-E1-Cre cells were consistently higher than those obtained with MDCK-E1 cells when co-infecting cells with high MOI of HV. Together, these observations show that MDCK-E1-Cre cells generate more non-infectious particles when using high MOI of virus.

When evaluating HV, no significant differences in PP:IP ratios were observed between the different MOIs or cell lines used (Table 2), probably because of the high interassay variability obtained when titrating infectious HV.

Discussion

While helper-dependent vectors (HDVs) continue to demonstrate a high therapeutic potential, the production standardization of these vectors is still limiting their wide availability in gene delivery assays and trials. In this study, we evaluated Cre-expressing cells stability, infection conditions and their relation to adenovirus amplification and helper vector (HV) contamination in serum-free medium con- ditions to establish a reproducible production process for HD canine adenovirus type 2 (CAV-2) vectors. We found that constitutive Cre expression affects the long-term capacity of cells to produce adeno- viruses, and co-infection conditions using low HV MOI can effec- tively reduce HV contamination even when using cells without Cre recombinase.

HDV producer cells need to be in culture for longer periods of time as cells are needed for serial amplifications until sufficient vec- tors are generated²,²⁵⁻²⁷. On the other hand, the expression of Cre recombinase, due to its toxicity⁷, is likely to impair cell line perform- ance. Therefore, the evaluation of producer cell line stability in the scope of HDV production becomes critical. The consistent MDCK- E1-Cre cells growth profile under increasing subculture passages (Figure 1) indicates that Cre expression was supported by the cell

line, probably because of non-toxic levels and/or the cells capa-
city to correct Cre-induced DNA double-strand breaks (DSBs)⁷.
However, when evaluating virus production, a consecutive reduction
in ΔE1 vectors (CAVGFP) productivity along culture passages was
observed (Figure 3), namely in the cells forced to express Cre recom-
binase through selective pressure from P10 to P20 (Figure 2).
MDCK-E1-Cre cells in Optipro SFM medium can be thus used for
virus production during 10 culture passages after thawing. The fac-
tor(s) and/or mechanism(s) behind these observations must be such
that despite impacting virus replication, cell homeostasis is balanced
enough to sustain growth under the presence of Cre. Furthermore,
any effect of E1 expression levels on CAV-2 production should be
ruled out, since E1 levels between MDCK-E1-Cre and MDCK-E1
cells in same conditions (passage number and antibiotic pressure)
were found to be similar (data not shown). The 116 cell line, one of
the several Cre-expressing cell lines derived from HEK 293²⁸ and
established by Palmer and Ng²⁶, was able to keep adenovirus pro-
ductivities up to 86 passages when maintained under selective pres-
sure. While these results differ from ours, the evaluation of other Cre-
expressing cells established for HDV production²⁸ should be pursued
to understand how broad/specific our results with MDCK-based cells
are and how host cell/cell clone can also contribute to the impact that
Cre has on the resulting cell performance.

The best infection conditions for HDV production must be well established as the MOI ratio of HDV/HV impacts the contamination levels of HV in co-infections. Similarly to what was shown with human HDV production²⁹, the best strategy to produce HD CAV-2 was under co-infections of 5 HDV: 0.1 HV, where maximum HDV productivity were ensured and HV contamination the lowest (Figure 4). Indeed, a low HV MOI can benefit HDV production by reducing the levels of excised helper DNA molecules after Cre recombinase activity, that are known to contribute to recombination between viral vectors³⁰, and circumventing the need to develop alternative strategies to attain higher levels of Cre than those supported by the cell line⁶. The relation between MOI ratio and HV contamination levels, namely that obtained in co-infections using MDCK-E1 cells (Figure 4B, D), indicates that MOI ratio is an effective strategy to unbalance the competition for amplification of HV in relation to HDV or ΔE1. In fact, this led to contamination levels as low as those obtained with MDCK-E1-Cre cells when producing HDV (Figure 4B), raising the possibility of producing HDV without the need of a recombinase when optimal MOI ratios are used. Therefore, as soon as HDV titers permit the establishment of such MOI ratio (usually after rescue step and 1st amplification), the subsequent amplifications can be moved to cell lines without Cre. This would lead to better vector yields, surpassing the negative effects that Cre has on cell performance⁷, recombination between vectors genomes³⁰ and virus amplification (Figure 3).

The analysis of PP:IP ratios also indicated that more non-
infectious particles were generated when using MDCK-E1-Cre cells
as producer cells, especially when co-infections were performed with
high MOI of HV (Figure 6). Although the differences in PP:IP ratio
between MDCK-E1 and MDCK-E1-Cre cells were rather low (up
to 1.6-fold differences), they were statistically significant under
high MOI of HV. More importantly, given that the PP:IP ratio of
clinical grade adenovirus vector is limited by the Food and Drug
Administration, special attention must be paid to this. These findings
substantiate the need to minimize the use of Cre recombinase
(expressing cells) and lower HV MOI to maximize HDV quality.
Following these observations, cell physiological state imposed by
the expression of Cre recombinase, probably aggravated under high
HV MOI, may be compromising the success of virus replication cycle
and virus particle maturation process. The relation between Cre
activity, infection conditions and virus maturation should be thus
further clarified to understand the mechanism behind the generation
of less infectious particles.
<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>Figure 4 | Effect of MOI ratio on virus production and HV contaminant using MDCK-E1-Cre and MDCK-E1 cells. (A, B) Cell specific productivity of HDV (A) and resulting HV contaminant (B). (C, D) Cell specific productivity of ΔE1 (C) and resulting HV contaminant (D). Values are shown as average ± standard-deviation (n = 3). * *p* < 0.05, ** *p* < 0.005, given by a single factor Anova analysis.</figcaption></figure>

Apart of generating more non-infectious particles under high virus MOI (Figure 6) and reducing virus production at high passage number (Figure 3), MDCK-E1-Cre cells also impacted CAV-2 pro- duction when co-infected with HV. In fact, when comparing the specific productivity of MDCK-E1 and MDCK-E1-Cre cells in ΔE1 + HV co-infections, a reduction in ΔE1 amplification was observed with MDCK-E1-Cre cells, specially under high HV MOI (Figure 4C). This feature is specific to co-infections with HV in MDCK-E1-Cre cells as this was not observed after infecting MDCK-E1-Cre and MDCK-E1 with increasing MOIs (from 5 to 40) of ΔE1 alone (Figure 3 and data not shown). Since the difference between co- infections with MDCK-E1-Cre and MDCK-E1 cells is the occurrence of HV genomes excision, these observations substantiate a relation between high number of excised HV genomes and inhibition of ΔE1 amplification.

To support Cre recombinase activity, cells must present a consist-
ent DSBs repair system. Such mechanism is involved in the inhibition
of adenovirus replication: viral genomes are sensed as DSBs, activ-

ating DNA damage response and the repair mechanism concatenate
viral genomes making them unavailable for the subsequent replica-
tion process³¹. Adenoviral E4 11 k (E4orf3) or the complex of E4 34 k
(E4orf6) and E1B proteins counteract and are sufficient to inhibit
DSBs repair system, thereby preventing genome concatenation³²⁻³⁵.
Given the link of Cre – DSBs repair – virus amplification, we hypo-
thesize that the reduction in CAV-2 production could be due to an up
regulation of DSBs repair mechanism. Assuming that Cre-expressing
cells should have an up regulated DSBs repair system, MDCK-E1-Cre
cells are likely to be more prone in sensing viral DNA as DSBs than
MDCK-E1 cells. If MDCK-E1-Cre cells are challenged with high
MOIs of HV, more DNA breaks are exposed as more viral genomes
are being excised, which might result in a higher activation of DSBs
repair system, explaining the inhibition of ΔE1 propagation under
high HV MOIs (Figure 4C). Accordingly, MDCK-E1-Cre cells with
increased DSBs repair system, being more prone to repair Cre-
induced damages, might dominate cell population during culture
passages due to proliferative advantage. Therefore, the DSBs repair
<figure><img src="image_12.png" /><figcaption>**Figure 5** | Effect of MOI on HV production in MDCK-E1-Cre and MDCK-E1 cells. Values are shown as average ± standard-deviation (n = 3).</figcaption></figure>

system of cells maintained in culture for long periods would be con-
sistently more active and sensitive to viral genomes, which might
explain the subsequent reduction of ΔE1 production in MDCK-E1-
Cre cells along passages (Figure 3). A model where DSB repair system
interfere with the typical adenovirus propagation must fit with a
scenario which E4 levels attained during infection are no longer
enough to inhibit this mechanism³²⁻³⁴. In addition, the occurrence
of genome concatenation should be observed to confirm these
assumptions³¹.

In conclusion, despite similar growth profile of MDCK-E1-Cre cells
with increasing passages, the long-term Cre recombinase expression
reduced their capacity to produce adenoviruses. Not only that, but
more non-infectious particles were generated with these cells (up to
1.6 fold-increase) in HV co-infections under high MOIs. The best
conditions to produce HDV followed a high HDV/HV MOI ratio
with a HV MOI as low as 0.1. This favored HDV propagation and
infectivity and limited the generation of contaminant HV. In fact, such
MOI ratio was capable of attaining low HV contamination levels, with
or without the activity of Cre, raising the possibility of producing
HDVs without Cre-expressing cells when optimal infection conditions

are used. This work unveils the impact of the Cre recombinase system
and MOI ratio on adenovirus propagation, highlighting important
bioprocess parameters that must be considered to design an improved
manufacturing of HDVs.

**Methods**

**Cell lines and culture media.** MDCK-E1 cells¹² are MDCK (ECCAC 841211903) derived and stably express the E1 region from CAV-2 and the neomycin resistance gene. MDCK-E1-Cre cells¹², in addition to E1, also express Cre recombinase and zeocin resistance gene. MDCK-E1 and MDCK-E1-Cre were sequentially adapted to Optipro serum-free medium (Gibco, Paisley, UK) with 4 mM glutamax (Gibco) by increasing the percentage of Optipro medium in subculture passages. Cells were subcultured twice a week in 150 cm² t-flasks and maintained in an incubator with humidified atmosphere of 5% CO₂ in air at 37°C. For splitting or collecting cells, the monolayer was washed with PBS, incubated with 0.25% (w/v) trypsin-EDTA (Gibco) at 37°C until detachment started to become evident (up to 15 min) and cells suspended in culture medium. Cells were then pelleted at 300 g during 10 min and re-suspended in fresh medium. To evaluate the effect of culture passages, cells were maintained in duplicates, either with or without antibiotic pressure. For antibiotic pressure, 500 µg/mL of Geneticin (G418) (Invivogen, Toulouse, France) was added to culture medium for MDCK-E1 cells maintenance, while for MDCK-E1-Cre cells G418 plus 500 µg/mL of Zeocin (Invivogen) was used. Cells were frozen once adapted to Optipro medium (P0) and after 10 and 20 cell culture passages in CryoStor CS10 (Sigma-Aldrich, St. Louis, MO, USA) using a Mr. Frosty Freezing Container (Thermo-scientific, Rockford, IL, USA).

**Viral vectors.** CAVGFP, JBΔ5 and HD CAVGFP are vectors derived from CAV-2 strain Toronto A 26/61, GenBank J04368. CAVGFP¹³ and HD CAVGFP¹⁶ are E1-deleted (ΔE1) and HD vectors, respectively, and contain an eGFP expression cassette. JBΔ5 is a helper vector (HV) containing loxP sites flanking the packaging domain and a RSV-lacZ expression cassette¹⁶. CAVGFP, JBΔ5 and HD CAVGFP viral stocks were prepared and purified by CsCl gradients as described previously¹²,¹³.

**Cell growth assays.** MDCK-E1-Cre growth assays in static cultures were performed using an inoculum of $1 \times 10^4$ cells/cm² in 25 cm² t-flasks with a working volume of 10 mL. Cell concentration and viability was determined by the trypan blue exclusion method using a 0.1% (v/v) solution prepared in PBS and counting cells in a Fuchs-Rosenthal haemacytometer (Brand, Wertheim, Germany).

**Cre activity.** Relative Cre activity was assayed by the level of luciferase activity generated by the adenovirus vector AdMA19 (a gift from F. Graham) after infecting cells³⁶. AdMA19 contains the luciferase cDNA under control of the human cytomegalovirus promoter but separated from it by an extraneous spacer sequence composed by a series of initiation and stop codons in several reading frames. This translational "start/stop sequence", flanked by loxP, disrupts translation. If the cell clone expresses Cre, the stop sequence is floxed out and translation proceeds leading to luciferase expression. Briefly, cells were seeded in 24 well-plates in quadruplicates and infected the day after with 20 IP/cell of AdM19 with medium exchange. Twenty-four hpi, cells were counted and lysed to release luciferase, both in duplicates. The

<figure><img src="image_13.png" /><figcaption>**Figure 6** | Comparison of physical to infectious particles ratio for HDV (A) and ΔE1 (B) obtained with MDCK-E1 and MDCK-E1-Cre cells in the different co-infections. Values are shown as average ± standard-deviation (n = 3). \* *p* < 0.05, given by a single factor Anova analysis.</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>Table 2 | Physical to infectious particles ratio of HV obtained with MDCK-E1-Cre and MDCK-E1 cells under the different co-infections</figcaption></figure>

<table><thead><tr><th rowspan="2">Co-infection with</th><th rowspan="2">Cells</th><th colspan="4">MOI of HV</th></tr><tr><th>5 HV</th><th>1HV</th><th>0.5 HV</th><th>0.1HV</th></tr></thead><tbody><tr><td rowspan="2">HDV</td><td>MDCK-E1-Cre</td><td>n.d.</td><td>86 ± 12</td><td>93 ± 17</td><td>85 ± 16</td></tr><tr><td>MDCK-E1</td><td>n.d.</td><td>75 ± 18</td><td>102 ± 33</td><td>93 ± 16</td></tr><tr><td rowspan="2">ΔE1</td><td>MDCK-E1-Cre</td><td>58 ± 8</td><td>50 ± 9</td><td>42 ± 3</td><td>n.d.</td></tr><tr><td>MDCK-E1</td><td>37 ± 19</td><td>38 ± 15</td><td>37 ± 7</td><td>n.d.</td></tr></tbody></table>

Values are shown as average ± standard-deviation (n = 3); No statistical mean (p value < 0.05) was obtained when comparing values of MDCK-E1-Cre with MDCK-E1 cells under same co-infection conditions. n.d.: not determined.

resulting supernatant was collected and the light units were quantified, with a Modulus Luminometer from Turner Biosystems (Sunnyvale, CA, USA) after adding luciferin, and normalized to cell concentration.

**Viral vectors production assays.** Cells were seeded at $3 \times 10^4$ cells/cm$^2$, infected the day after with medium exchange. To evaluate cells productivity at different passage number, infections of CAVGFP at MOI 5 were performed. Excision efficiency was estimated by determining the fold-reduction in helper JBA5 production with MDCK-E1-Cre cells infected at MOI 5. The assays for HD CAVGFP production optimization were performed by co-infecting cells with HD CAVGFP at MOI 5 and JBA5 at MOI 1, 0.5 and 0.1. To further understand how Cre recombinase and co-infection determine optimal HD CAVGFP production, co-infections of CAVGFP (MOI 5) with JBA5 (MOI 5, 1 and 0.5), and single infections of JBA5 (MOI 5, 1 and 0.5) were also performed. Moreover, all the infection assays were performed in MDCK-E1-Cre and MDCK-E1 cells. Once infected, cells were incubated for further 48 h and viral samples collected using triton (Sigma-Aldrich) 0.1% (v/v) in Tris-HCl, clarified at 3000 g for 10 min at 4°C and stored at -85°C until further analysis.

**Infectious vectors titration.** Quantification of infectious CAVGFP and HD CAVGFP was performed by monitoring the expression of GFP, while titration of infectious JBA5 vectors was based in lacZ gene expression and β-galactosidase activity as described previously¹².

**Quantification of physical particles.** Viral genomes were extracted and purified by High Pure Viral Nucleic Acid Kit (Roche Diagnostics, Penzberg, Germany). SYBR Green I dye chemistry was used to detect PCR products using Lightcycler system. Previously purified and quantified plasmids coding for E1-deleted CAV-2 genome, GFP or lacZ were used as standards. To ensure the removal of free viral genomes from samples and accurately quantify viral physical particles, a benzonase treatment was performed prior to DNA extraction by adding 2 µl of benzonase (250 U/µl) (Merck Millipore, Darmstadt, Germany) to the same sample volume and incubating for 1 h at 37°C. Benzonase was inhibited during the proteinase K step of DNA extraction protocol. To estimate the number of viral genomes, primers against the expression cassette of each viral vector were used. Primers for GFP gene: forward 5'-CAGAGA-AGAACGGCATCAAGGT-3' and reverse 5'-CTGGGTGCTCAGGTAGTGG-3'; primers for lacZ gene: forward 5'-ACTATCCCGACCGCCTTACT-3' and reverse 5'-TAGCGGCTGATGTTGAACTG-3'.

1. Dormond, E., Perrier, M. & Kamen, A. From the first to the third generation adenoviral vector: what parameters are governing the production yield? *Biotechnol Adv* 27, 133-144 (2009).

2. Parks, R. J. *et al.* A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal. *Proc Natl Acad Sci USA* 93, 13565-13570 (1996).

3. Lieber, A., He, C. Y., Kirillova, I. & Kay, M. A. Recombinant adenoviruses with large deletions generated by Cre-mediated excision exhibit different biological properties compared with first-generation vectors in vitro and in vivo. *J Virol* 70, 8944-8960 (1996).

4. Hardy, S., Kitamura, M., Harris-Stansil, T., Dai, Y. & Phipps, M. L. Construction of adenovirus vectors through Cre-lox recombination. *J Virol* 71, 1842-1849 (1997).

5. Ng, P., Evelegh, C., Cummings, D. & Graham, F. L. Cre levels limit packaging signal excision efficiency in the Cre/loxP helper-dependent adenoviral vector system. *J Virol* 76, 4181-4189 (2002).

6. Gonzalez-Aparicio, M. *et al.* Self-inactivating helper virus for the production of high-capacity adenoviral vectors. *Gene Ther* 18, 1025-1033, doi:10.1038/gt.2011.58 (2011).

7. Loonstra, A. *et al.* Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells. *Proc Natl Acad Sci USA* 98, 9209-9214, doi:10.1073/pnas.161269798 (2001).

8. Thyagarajan, B., Guimaraes, M. J., Groth, A. C. & Calos, M. P. Mammalian genomes contain active recombinase recognition sites. *Gene* 244, 47-54 (2000).

9. Silver, D. P. & Livingston, D. M. Self-excising retroviral vectors encoding the Cre recombinase overcome Cre-mediated cellular toxicity. *Molecular cell* 8, 233-243 (2001).

10. Pfeifer, A., Brandon, E. P., Kootstra, N., Gage, F. H. & Verma, I. M. Delivery of the Cre recombinase by a self-deleting lentiviral vector: efficient gene targeting in vivo. *Proc Natl Acad Sci USA* 98, 11450-11455, doi:10.1073/pnas.201415498 (2001).

11. Baba, Y., Nakano, M., Yamada, Y., Saito, I. & Kanegae, Y. Practical range of effective dose for Cre recombinase-expressing recombinant adenovirus without cell toxicity in mammalian cells. *Microbiol Immunol* 49, 559-570 (2005).

12. Fernandes, P. *et al.* Impact of E1 and Cre on adenovirus vector amplification: developing MDCK CAV-2-E1 and E1-Cre transcomplementing cell lines. *PLoS One* 8, e60342, doi:10.1371/journal.pone.0060342 (2013).

13. Kremer, E. J., Boutin, S., Chillon, M. & Danos, O. Canine adenovirus vectors: an alternative for adenovirus-mediated gene transfer. *J Virol* 74, 505-512 (2000).

14. Perreau, M. *et al.* Contrasting effects of human, canine, and hybrid adenovirus vectors on the phenotypical and functional maturation of human dendritic cells: implications for clinical efficacy. *J Virol* 81, 3272-3284, doi:10.1128/JVI.01530-06 (2007).

15. Soudais, C., Laplace-Builhe, C., Kissa, K. & Kremer, E. J. Preferential transduction of neurons by canine adenovirus vectors and their efficient retrograde transport in vivo. *Faseb J* 15, 2283-2285 (2001).

16. Soudais, C., Skander, N. & Kremer, E. J. Long-term in vivo transduction of neurons throughout the rat CNS using novel helper-dependent CAV-2 vectors. *FASEB J* 18, 391-393, doi:10.1096/fj.03-0438fje (2004).

17. Hnasko, T. S. *et al.* Cre recombinase-mediated restoration of nigrostriatal dopamine in dopamine-deficient mice reverses hypophagia and bradykinesia. *Proc Natl Acad Sci USA* 103, 8858-8863, doi:10.1073/pnas.0603081103 (2006).

18. Pivetta, C., Esposito, M. S., Sigrist, M. & Arber, S. Motor-circuit communication matrix from spinal cord to brainstem neurons revealed by developmental origin. *Cell* 156, 537-548, doi:10.1016/j.cell.2013.12.014 (2014).

19. Ekstrand, M. I. *et al.* Molecular profiling of neurons based on connectivity. *Cell* 157, 1230-1242, doi:10.1016/j.cell.2014.03.059 (2014).

20. Ariza, L. *et al.* Central Nervous System Delivery of Helper-Dependent Canine Adenovirus Corrects Neuropathology and Behavior in Mucopolysaccharidosis Type VII Mice. *Hum Gene Ther* 25, 199-211, doi:10.1089/hum.2013.152 (2014).

21. Cubizolle, A. *et al.* Corrective GUSB Transfer to the Canine Mucopolysaccharidosis VII Brain. *Mol Ther* 22, 762-773, doi:10.1038/mt.2013.283 (2014).

22. Serratrice, N. *et al.* Corrective GUSB transfer to the canine mucopolysaccharidosis VII cornea using a helper-dependent canine adenovirus vector. *Journal of controlled release: official journal of the Controlled Release Society* 181, 22-31, doi:10.1016/j.jconrel.2014.02.022 (2014).

23. Fernandes, P. *et al.* Bioprocess development for canine adenovirus type 2 vectors. *Gene Ther* 20, 353-360, doi:10.1038/gt.2012.52 (2013).

24. Fernandes, P. *et al.* Impact of Adenovirus life cycle on the generation of canine helper-dependent vectors. *Gene Ther* 22, 40-49, doi:10.1038/gt.2014.92 (2015).

25. Dormond, E. *et al.* An efficient and scalable process for helper-dependent adenoviral vector production using polyethylenimine-adenofection. *Biotechnol Bioeng* 102, 800-810, doi:10.1002/bit.22113 (2009).

26. Palmer, D. & Ng, P. Improved system for helper-dependent adenoviral vector production. *Mol Ther* 8, 846-852 (2003).

27. Suzuki, M. *et al.* Large-scale production of high-quality helper-dependent adenoviral vectors using adherent cells in cell factories. *Hum Gene Ther* 21, 120-126, doi:10.1089/hum.2009.096 (2010).

28. Kovesdi, I. & Hedley, S. J. Adenoviral producer cells. *Viruses* 2, 1681-1703, doi:10.3390/v2081681 (2010).

29. Dormond, E., Perrier, M. & Kamen, A. Identification of critical infection parameters to control helper-dependent adenoviral vector production. *J Biotechnol* 142, 142-150, doi:10.1016/j.jbiotec.2009.03.021 (2009).

30. Ahn, M. *et al.* Vector and helper genome rearrangements occur during production of helper-dependent adenoviral vectors. *Human gene therapy methods* 24, 1-10, doi:10.1089/hgtb.2012.198 (2013).

31. Weiden, M. D. & Ginsberg, H. S. Deletion of the E4 region of the genome produces adenovirus DNA concatemers. *Proc Natl Acad Sci USA* 91, 153-157 (1994).

32. Boyer, J., Rohleder, K. & Ketner, G. Adenovirus E4 34 k and E4 11 k inhibit double strand break repair and are physically associated with the cellular DNA-dependent protein kinase. *Virology* 263, 307-312, doi:10.1006/viro.1999.9866 (1999).
<figure><img src="image_15.png" /></figure>

33. Stracker, T. H., Carson, C. T. & Weitzman, M. D. Adenovirus oncoproteins inactivate the Mre11-Rad50-NBS1 DNA repair complex. *Nature* **418**, 348–352, doi:10.1038/nature00863 (2002).

34. Araujo, F. D., Stracker, T. H., Carson, C. T., Lee, D. V. & Weitzman, M. D. Adenovirus type 5 E4orf3 protein targets the Mre11 complex to cytoplasmic aggresomes. *J Virol* **79**, 11382–11391, doi:10.1128/JVI.79.17.11382-11391.2005 (2005).

35. Baker, A., Rohleder, K. J., Hanakahi, L. A. & Ketner, G. Adenovirus E4 34 k and E1b 55 k oncoproteins target host DNA ligase IV for proteasomal degradation. *J Virol* **81**, 7034–7040, doi:10.1128/JVI.00029-07 (2007).

36. Soudais, C., Boutin, S. & Kremer, E. J. Characterization of cis-acting sequences involved in canine adenovirus packaging. *Mol Ther* **3**, 631–640 (2001).

## Acknowledgments

We thank Sandy Ibanes and Aurelie Gennetier for the initial preparations of CAV-2 vectors. This work was supported by Fundação para a Ciência e a Tecnologia (FCT) – Portugal, through the project PTDC/EBB-BIO/118615/2010, and European Commission through FP7 project BrainCAV (222992). Paulo Fernandes acknowledges FCT for his Ph.D. grant (SFRH/BD/70810/2010).

## Author contributions

P.F. designed the experiments; P.F. and A.I.A. performed the experiments; P.F. analyzed the data; P.F. and A.S.C. wrote the manuscript; E.J.K., P.M.A. and A.S.C. contributed with materials, equipments and analysis tools; all authors discussed the results and reviewed the manuscript.

## Additional information

Competing financial interests: The authors declare no competing financial interests.

How to cite this article: Fernandes, P., Almeida, A.I., Kremer, E.J., Alves, P.M. & Coroadinha, A.S. Canine helper-dependent vectors production: implications of Cre activity and co-infection on adenovirus propagation. *Sci. Rep.* **5**, 9135; DOI:10.1038/srep09135 (2015).

<figure><img src="image_16.png" /></figure>

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder in order to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/