Reconfiguration of AAV Rep-Cap coding sequences significantly increases viral vector yield

and enables scalable production in suspension HEK-293 cells

A. Bray, A. Gillman, C. Branciaroli, W. Su, T. Payne, R. Cawood
OXGENE | Medawar Centre | Robert Robinson Avenue | Oxford | OX4 4HG | United Kingdom | www.oxgene.com
Corresponding author: abray@oxgene.com | For information about our technologies, please contact us at business@oxgene.com

Introduction Manipulation of Ad5 helper genes

rAAV vectors are demonstrating significant clinical benefit in patients for a range of diseases, however the Many available adenoviral helper plasmids commercially available contain segments of adenoviral DNA that
ability to produce large quantities of functional rAAV particles in a scalable manufacturing system remains do not correspond to the known AAV helper functions. We compared four of these helper plasmids to our own,
challenging. The majority of current rAAV production systems exploit the native Rep-Cap expression smaller, helper plasmid, pSF-E2A-E4-VAI. The latter was specifically cloned to exclude additional Ad5
configuration found in the viral genome, and adenovirus helper plasmids which contain non-essential regions sequences. We compared the rAAV viral titres produced by qPCR to assess if our pared back plasmid
due to historical cloning strategies. OXGENE has explored the re-design of these two aspects at the DNA level, performed similarly to the larger helper plasmids.
to increase the yield and quality of rAAV vectors produced. Not only is this system superior to industry standard Helper plasmid Plasmid size (bp)
Aligned regions of adenovirus
type 5 genome◊
plasmids in transient transfection in Adherent 293T cells, it is superior in suspension HEK293 cells which allows ns
10,435 - 11,160
1×10 11 E2A-E4-VAI 11275 22,118 - 27,534
for scalable production. ns 32,636 - 35,836
9840 - 13,258
Helper 1 15376 21,443 - 28,134

VG/mL
30,820 - 35,813
9841 - 10,949
Reconfiguration of native Rep-Cap coding sequences
1×10 10
Helper 2 17867 21,878 - 28,137
30,819 - 35,937
9842 - 13,258
We developed a number of re-configured Rep-Cap plasmids using heterologous promoters and/or an Internal Helper 3 18879 21,443 - 28,137
30,819 - 35,937
1×10 9
Ribosome Entry Site (IRES) to drive the Rep and Cap coding regions. We chose to express the Rep sequence at 9855 - 11,548

Helper 1

Helper 2

Helper 3
E2A-E4-VAI

Helper 4*
Helper 4 21834 21,546 - 28,598
a lower level due to well-documented cytotoxic and cytostatic effects. We measured the ability of the resulting 30,728 - 35,938
◊ Accession number: AY339865
packaging plasmids to influence rAAV genomic titre by qPCR, and by ELISA to compare viral particle titre.
Table 1. Helper plasmids compared by plasmid size (bp) and
Helper plasmid
alignment to Ad5 reference sequence AY339865

(A) Figure 3. Comparison of rAAV5 production using helper plasmids in E125 flask HEK293_BCD_SP7D5 suspension cell
production. Error bars depict 95% CI. Dunn's multiple comparisons test found no significant differences between pSF-E2A-E4-
VAI and the top two performing commercial helper plasmids.
*Helper 4 plasmid contains RepCap5 sequences, and therefore pSF-RepCap5 was not supplied during production.

Additional Ad5 sequences do not confer additional rAAV titre, therefore we next sequentially removed coding
sequences from pSF-E2A-E4-VAI to generate a smaller, novel helper plasmid.

E2-L L4P E2P E4P

E4
r

Figure 4 (left). pSF-E2A-E4-VA

schematic indicating adenoviral
Transcript Gene Product genes encoded within. Coloured
1×10 12 E2A DBP
(B) (C) 1×10 12 genes are known to be required
✱✱✱ L4 100K

✱✱✱✱
L4 33K for rAAV production. Internal
ORF6
L4 22K
adenoviral promoters are shown
1×10 11 ✱✱✱✱ E4 ORF1
1×10 11
above the plasmid map. The
VG/mL

VP/mL

VA RNA I
E4 ORFB
E4 ORF3 VA RNA II table lists the transcripts on
1×10 10
E4 ORF4
which each gene is typically
1×10 10 E4 ORF6
E4 34K found.
E2B VA RNA I
E2B VA RNA II
1×10 9
1×10 9
a b c d e f g
a b c d e f g
Packaging Plasmid
ns
Packaging Plasmid (A)
Figure 5 (right and below). Comparison of 1×10 11

(D) 40 sequentially truncated helper plasmids in 24-deep

Figure 1. Comparison of rAAV5 production using re-configured
32.04

well plate HEK293_BCD_SP7D5 suspension cell

VG/mL

packaging plasmids in 24-deep well plate

27.96

27.85
% full particles

1×10 10
30 production. (A) Viral genome titres produced with
HEK293_BCD_SP7D5 suspension cell production. (A)
21.88

sequentially truncated helper plasmid, or CMV-

19.62

schematics of packaging plasmids tested, (B) viral genomes per

expressed Ad5 coding sequences, (B) viral genome
16.18

20
mL produced using each plasmid tested, (C) viral particles per 1×10 9
titres when comparing full E2A region (blue) with
mL produced using each plasmid tested, (D) percentage full
10 CMV-DBP (red) when supplied in trans. (C) viral
6.28

particles calculated using the mean VP/mL and mean VG/mL for
I

E4
I

f6
I

4K
A
A

genome titres between single helper plasmids with

or
-V
V

-V

_3
4-

E4
E4

each plasmid tested. Error bars depict 95% CI. Dunn's multiple
f6
-E

34

V-

either full E2A region (as OXGENE standard) or

or
A

0
M
E2

E4
f6

comparisons test carried out on data from (B) and (C), ***
or

V-

CMV-E2A_DBP supplied in cis. Error bars depict

E4

a b c d e f g
M
C
V-

p<0.001, **** p<0.0001, n=12

M

Packaging Plasmid 95% CI. Dunn's multiple comparisons test carried

C

Helper plasmid
out on data, **** p<0.0001. No significant
differences between combinations tested in (B).

Re-configured packaging plasmid verification in multiple serotypes 1×10 11 4×10 10

✱✱✱✱

(B) (C)
3×10 10
We chose one of the top performing packaging plasmid designs (pSF-CMV-Cap-EMCV-Rep) and cloned in each
VG/mL
VG/mL

of the capsid serotypes 1-9. We compared the ability of each of these to produce rAAV compared to the standard 1×10 10 2×10 10

configuration. We analysed the resulting virus by qPCR, ELISA and TCID50. 1×10 10

1×10 9 0
(A) (B)
P
P
P

A
A
I

I
A

25
A

A
B

B
B

E2
E2
E2
-V

20
Fold change in VP/mL

-V
_D
_D
_D
Fold change in VG/mL

4-
4

I+

+
I+

E4
-E

A
A
A

-E
K

E2
E2
A
E2
A
A

P-
34

A
-V
-V
E2

20
V-
V-

B
E2
V-

6_
4K
E4

_D
M

M
M

15
f
or
_3

C
C
C

A
E2
E4

+
I+
I+

f6

4K
or

15
V-
A

V-
A

-V
-V

E4

_3
M

M
4K
E4

C
V-

f6

10 Helper plasmid
_3

or
M
C

E4

10
f6
or

V-
E4

5
C
V-

5
M
C

Helper plasmid(s)
0 0
1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7* 8 9 Our data shows that larger helper plasmids including additional Ad5 genomic DNA does not increase rAAV viral
(C) Serotype Serotype titre. We further show that it may be possible to further reduce helper plasmid size by removing unnecessary
50 genes (Fig. 5B), however when we made a novel, smaller helper plasmid, we saw a 3-fold reduction in rAAV
Fold change in IU/mL

Figure 2. Comparison of rAAV production using re-configured genomic titre. This may be due to stoichiometric effects, and further experiments may confirm this hypothesis.
40
packaging plasmids compared to standard configuration, for
30
serotypes 1-9. Values given are fold-change of the means Conclusions
20 compared to the standard configuration (n=6) (A) fold change in
viral genomes measured by qPCR (B) fold change in viral We reconfigured the native AAV RepCap genes by placing the Cap and Rep expression cassettes under the
10
particles measured by ELISA. *No ELISA kit is available for
0 control of a CMV promoter and EMCV IRES sequences, respectively. This configuration shows higher viral
AAV4 or AAV 7. No measurement obtained for serotype 4,
1 2 3 4 5 6 7 8 9
however we used an AAV8 kit to measure serotype 7 due to genome titre across multiple serotypes and shows an increase in the percentage of packaged particles. We
Serotype homologous epitopes. (C) fold change in infectious units further validated our specifically designed adenoviral helper plasmid, showing that it produces the highest
measured by infectious titre assay in Hela-RC32 cell line. titres of rAAV. Our transient rAAV production system, together with our suspension HEK293_BCD_SP7D5
cell line allows scalable high titre and high quality rAAV production.