Potency Assay for AAV Vector Encoding Retinal Pigment Epithelial 65 Protein Linda Couto, PhD1, George Buchlis, PhD1, Rafal Farjo, PhD2, Katherine A. High, MD1

1Spark Therapeutics, Inc., Philadelphia, PA; 2EyeCRO, LLC, Oklahoma City, OK

Purpose • A validated potency assay for release of a medicinal product is required by the

FDA prior to commercialization. SPK-RPE65 is a gene transfer vector being developed for the treatment of inherited retinal degeneration due to mutations in the retinal pigment epithelial 65 (RPE65) gene. In a Phase 3 trial, it demonstrated restoration of functional vision in subjects progressing toward blindness. We developed a non-radioactive in vitro assay to measure the activity of SPK-RPE65, in anticipation of potential commercialization of this product.

References 1. Moiseyev et al, 2005. 2. Palczewski, 2006. 3. Ridge & Palczewski, 2007. 4. Jacobson et

al., 2005. 5. Takahashi et al., 2014.

Acknowledgements Irena Ignatova, PhD, Spark Therapeutics; Parveen Kaushal, PhD, VolPal, LLC;

Karen Doucette, Absorption Systems, Exton, PA; Wei Zhang, Absorption Systems, Exton, PA; Jennifer Nawn, Absorption Systems, Exton, PA; Dezhong Liu, Absorption Systems, Exton, PA.

Support This study was sponsored by Spark Therapeutics.

Disclosures Linda Couto, George Buchlis, Katherine A. High and Irena Ignatova are employees of Spark

Therapeutics.

Poster # C0048

Methods • The RPE65 gene encodes an isomerohydrolase that converts all-trans-retinol

to 11-cis-retinol, and is critical to the visual cycle. The potency assay is a modification of a radioactive assay (Moiseyev et al, 2005), and includes three components:

1. cell transduction by SPK-RPE65;

2. enzymatic assay measuring RPE65 activity; and

3. detection of the reaction product, 11-cis-retinol, by LC-MS/MS.

Results

Conclusions • A non-radioactive potency assay has been developed for SPK-RPE65. The

assay is useful for release and stability testing of the vector. In addition, it is being used to test the activity of many mutant RPE65 genes in order to confirm and uncover disease-causing variants.

Presented at the

Annual Meeting of the Association for

Research in Vision and Ophthalmology

May 1-5, 2016 | Seattle, Washington

Figure 1. Schematic showing the role of RPE65 in the visual cycle

characterization of the visual cycle is still debated [9,10]. The

evidence for an alternate pathway has been first evidenced in

cone-dominated chicken retinas [11,12]. Recently, DES1, a

trans- to cis-retinol isomerase enzyme expressed in the Muller

glial cells, has been identified and characterized [13].

For both visual cycles in vivo experiments report globally high

substrate specificity toward 11-cis-retinal as the active chromo-

phore. Specifically, past experimental determination of retinoid

isomers in the mouse eye has shown the presence of other cis-

forms (i.e. 9-cis and 13-cis) only in negligible quantities [13,14]

(Fig. 2). This established paradigm set the initial idea that the var-

ious proteins involved in the cyclic process are specific for either

11-cis or all-trans-retinoids, so that no other isomers are pro-

duced/transduced in chemically relevant concentrations in the

eye. However, independent experiments targeting single

components of the visual cycle have been depicting a more

complex framework.

Since several decades rhodopsin was shown to bind and to be

photochemically active in complex with retinaldehydesother than

11-cis. In particular, opsin bound to9-cis-retinal and9–13-dicis-ret-

inal (called isorhodopsin-I and II) were shown to provoke action

potentials in vivo in various animal models [15,16]. In more recent

times, distinct componentsof thevisual cycle(RPE65,RDH5,CRAL-

BP, DES1) were reported to exhibit specificity for alternate cis-con-

formations [6,13,17,18]. These findings include evidence for the

in vitro formation of relevant quantities of 13-cisand 9-cis-retinol,

at least comparable to those of 11-cis-retinol, by RPE65 [6]. Also

the recently identified DES1 isomerase produces in vitro and

in vivo mixtures of cis-retinols [13]. Other components in RPE, like

RDH5 and CRALBP,show catalytic activity and goodbindingaffinity

respectively for 9-cis-retinol/al substrates [17,18]. All these recent

findings suggest that either retinoids other than 11-cisor all-trans

may be effectively produced but efficiently processed in the eyeor

that in vivo co-expression and interaction of distinct visual cycle

components enhances selectivity towards11-cisconformation.

In this short review, we shall discuss recent progress on the

(bio)chemistry of retinoids in thevisual cycle. In particular wewill

address new findingsabout retinoid conformations other than 11-

cisor all-trans.Because of their better characterization, weshall fo-

cus mostly on proteins expressed in the rod-RPEcell system, with

some cone-Müller associated examples also being presented. This

review does not intend to be an exhaustive work about the visual

cycle and its components, or on retinoid (bio)chemistry. For more

extended analyses on these topics, reference to other works (for

example: [4,19–22]) may be envisaged.

General retinoid metabolism

Biological processing of retinoids requires multiple extra- and

intracellular binding proteinsable to recognizeand traffic their dif-

ferent chemical and isomeric forms. Evolution of retinoid-specific

binders isdriven by theneed for facilitating diffusion of poorly sol-

uble substrates and also by the necessity for sequestration of

Fig. 1. Visual cycle in the Rod-RPEsystem. Simplified scheme of the Rod-RPEvisual cycle. The main retinoid species entering into the cycle, as well as the principal protein

components responsible for their transformation, are reported.

Fig. 2. Chemical structures of the retinoids discussed in the text.

188 M. Cascella et al./Archives of Biochemistry and Biophysics 539 (2013) 187–195

all-trans-retinol

11-cis-retinol

11-cis-retinal

11-cis-retinal

All-trans-retinyl ester

all-trans-retinol

all-trans-retinal

CRBP

LRAT: lecithin:retinol acyltransferase; CRBP: cellular retinol binding protein (all-trans-retinol and all-trans-retinal; CRALBP:

cellular retinaldehyde binding protein; IRBP: interphotoreceptor retinol binding protein

Figure 1. The RPE65 gene encodes an isomerohydrolase, which is critical to the function of the retinoid cycle. Visual perception results from the biological conversion of light energy to electrical signaling by retinal photoreceptors in the eye. Eleven-cis retinal is a chromophore, which binds to apoprotein G protein-coupled receptor opsins (rhodopsin) in photoreceptors to form a visual pigment. Upon absorption of a photon, the 11-cis-retinal undergoes photoisomerization to its trans form (Palczewski, 2006; Ridge & Palczewski, 2007). The isomerized chromophore, all-trans-retinal, is reduced to all-trans-retinol, transported to the RPE, and converted to fatty acid all-trans-retinyl esters by lecithin/retinol acyltransferase (LRAT). In the presence of cellular retinaldehyde binding protein (CRALBP), RPE65 converts this ester to 11-cis-retinol, and regeneration of 11-cis-retinal by 11-cis-retinol dehydrogenase, completes the retinoid (visual) cycle. The biochemical blockade of the visual cycle resulting from RPE65 deficiency causes a profound impairment in visual function with delayed degeneration of retinal cells (Jacobson et al., 2005).

Figure 2. In vitro assay to demonstrate potency of AAV2-hRPE65v2

vectors

HEK293-LRAT cells (express lecithin:retinol acyltransferase)

SPK-RPE65

72 hrs

Lysate (100 mg)

• all-trans-retinol • CRALBP • 200 mL reaction vol

In dark, 2 hrs

Extract with 300 mL Hexane LC-MS/MS Detect and quantify 11-cis-retinol

1) Transduction of HEK293/LRAT cells with SPK-RPE65 2) Isomerohydrolase assay 3) Detection/quantification of 11-cis-retinol by LC-MS/MS

3 STEPS:

Figure 2. HEK293 cells constitutively expressing lecithin-retinol acyltransferase (LRAT) are transduced with SPK-RPE65 at different multiplicities of infection (MOI), ranging from 3x103–2x106 vector genomes (vg)/cell. Seventy-two hours later, cells are harvested, homogenized, and cell lysates containing 100 µg of total protein are used in the isomerohydrolase assay. Cell lysates are incubated at 37oC with all-trans-retinol and CRALBP for 2 hours in the dark. Following the incubation, the reaction is stopped by adding 300 µL MeOH and extracted with 300 µL hexane. The organic phase is removed, evaporated, resuspended in a reconstitution solution and analyzed by LC-MS/MS for 11-cis-retinol.

Compound name: 11-cis-retinol

Coefficient of Determination: R^2 = 0.995072

Calibration curve: 0.0407484 * x̂ 2 + 111.274 * x + -80.4315

Response type: External Std, Area

Curve type: 2nd Order, Origin: Exclude, Weighting: 1/x̂ 2, Axis trans: None

Conc-0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0

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min0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

0

MRM of 1 channel,API+

269.22 > 93.14

20151005_MD_a074

500 nM each: all-trans-/cis-retinol in Recon 20151006_sam

6.075e+00511-cis-retinol

6.10

6.55

All-trans-retinol

Good sensitivity and linearity

Good separation of substrate and product

% o

f la

rges

t p

eak

Figure 3. LC-MS/MS analysis of 11-cis-retinol

Extracted 11-cis-retinol STD curve (1-20 nM)

Figure 3. An LC-MS/MS method was developed for detection of 11-cis-retinol using a Waters Xevo TQ-S instrument. Separation of 11-cis-retinol from all-trans-retinol, 9-cis-retinol, 13-cis-retinol, and other retinoids has been achieved with an 11-cis-retinol calibration range of at least 1 nM – 20 nM.

Figure 4. Optimization of isomerohydrolase assay conditions

Optimized conditions: 100 mg cell protein

1 mM all-trans-retinol 10 mM CRALBP

min0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50

%

11

MRM of 1 channel,API+

269.22 > 93.14

20151005_MD_a043

Sample 20151006: #8-1 20151006_sam

1.732e+00411-cis-retinol

6.10

2.24

1.991.830.700.11 1.23

4.76

3.392.33

2.85

3.66

4.383.95

5.514.93

7.55

6.55

6.226.81

7.13

*

CRALBP (mM)

atROL (mM)

5 10 25 5 10 25

0.2

5 10 25

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ino

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Assay Design: HEK293/LRAT cells transduced with SPK-RPE65 (1x105 vector

genomes/cell) Evaluate enzymatic assay by varying substrate and CRALBP

reagents

Figure 4. HEK293/LRAT cells were transduced with SPK-RPE65 at an MOI of 1x105 vg/cell. Cell lysates (100 µg total protein) were incubated with varying concentrations of all-trans-retinol (0.2, 0.4, 1.0 µM) and varying concentrations of CRALBP (5, 10, 25 µM), and the amount of 11-cis-retinol produced was compared. The concentration of 11-cis-retinol increased as the substrate concentration increased, but the concentration of CRALBP did not impact 11-cis-retinol levels.

Figure 5. Potency assay is reproducible

13.8 nM +/-0.73 CV= 5.3%

0.0

2.0

4.0

6.0

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10.0

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Transduction RPE65 assay

1 2 3

A B C A B C A B C

11

-cis

-ret

ino

l (n

M)

Assay Design: HEK293/LRAT cells transduced with SPK-RPE65 (1x105 vector genomes/cell) Three independent transductions Cell lysates analyzed for RPE65 activity (3 assays/lysate)

Figure 5. To assess the reproducibility of the assay, HEK293/LRAT cells were transduced in triplicate with SPK-RPE65 (MOI=1x105 vg/cell), and each cell lysate was analyzed in three separate enzymatic assays. In the example shown here, the average level of 11-cis-retinol produced from these nine assays was 13.8 nM +/- 0.7 (CV=5.3%), demonstrating good intra-assay reproducibility.

11

-cis

-ret

ino

l (n

M)

Log MOI

Assay Design: HEK293/LRAT cells transduced with SPK-RPE65 vectors 8 MOIs (3x103, 1x104, 3x104, 1x105, 2.5x105, 5x105, 1x106, 2x106 vector genomes/cell) Three independent transductions Cell lysates analyzed for RPE65 activity (3 assays/lysate)

Figure 7. Use of assay to measure relative potency of vectors

Statistical tests for parallelism

Probability

F test 0.643

ChiSquare 0.895

Conclusion: The Prob>F and Prob>ChiSq (p-values) are both >0.05, indicating that there are no differences between the curves. Suggests two lots of vectors are of similar potency.

Assay Design: HEK293/LRAT cells transduced with two different lots of SPK-RPE65 Five MOIs (3x103, 1x104, 3x104, 1x105, 3x105 vector genomes/cell) Cell lysates analyzed for RPE65 activity

4 Parameter Logistic Model used to compare two lots of vector

Figure 7. This assay is being developed as a release test for SPK-RPE65. As such, it is important for the assay to measure relative potency of multiple vector lots. HEK293/LRAT cells were transduced with two different lots of SPK-RPE65 at five MOIs ranging from 3x103–3x105 vg/cell, in triplicate. Cell lysates were analyzed in triplicate by incubation with all-trans-retinol and CRALBP, and the amount of 11-cis-retinol produced was measured by LC-MS/MS. For both lots of vector analyzed, the plots of 11-cis-retinol as a function of MOI form two parallel sigmoidal curves. Statistical testing for parallelism concluded that the two vectors have similar potency.

Figure 8. Use of assay to determine activity of RPE65 variants

fg R

PE6

5/G

AP

DH

(rel

to

WT)

0.00

50.00

100.00

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250.00

300.00

4 u

g

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ug

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RPE65

Actin

E20delG V473D E254D

RPE65

Actin

rhRPE65

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ug

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rhRPE65

RPE65 mRNA Levels

RPE65 Protein Levels

RPE65 Variant Mutation

WT

Human/Chicken hybrid (sIMH)

S2Y,I3S,L26V,N170K,K297G

Variant 1 E20delG

Variant 2 V473D

Variant 3 E254D

Figure 8. The assay can be used to assess the biological activity of RPE65 variants. As a proof-of-concept, AAV2 vectors encoding five different RPE65 genes were analyzed in the assay. HEK293/LRAT cells were transduced in duplicate with AAV2 vectors (MOI=1x105 vg/cell) encoding different RPE65 variants: SPK-hRPE65, AAV2-RPE65 (chicken/human hybrid; sIMD) (Takahashi et al, 2014), AAV2-hRPE65(E20delG), AAV2-hRPE65(V473D) and AAV2-hRPE65(E254D). (A) RPE65 mRNA levels were measured by a quantitative PCR assay. (B) RPE65 protein levels were measured by western blot (25 µg total protein loaded). (C) Isomerohydrolase activity was measured by incubating 100 µg total protein with all-trans-retinol (1.0 µM) and CRALBP (25 µM), and 11-cis-retinol production was detected by LC-MS/MS. All five AAV-RPE65 variant vectors expressed RPE65 mRNA, but three of the variants did not express RPE65 protein, and thus did not generate 11-cis-retinol in the enzymatic assay. AAV2-hRPE65 and AAV2-RPE65 (chicken/human hybrid; sIMD) expressed both RPE65 mRNA and protein and as predicted, the latter generated more 11-cis-retinol than the wild-type RPE65.

0

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-cis

-ret

ino

l (n

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Isomerohydrolase Levels

MOI Ave SD CV

3.00E+03 BLOQ

1.00E+04 BLOQ

3.00E+04 2.2 0.436 20.11%

1.00E+05 4.2 0.944 22.33%

2.50E+05 6.6 1.219 18.43%

5.00E+05 9.0 1.218 13.47%

1.00E+06 10.7 0.756 7.04%

2.00E+06 9.9 1.386 14.02%

Figure 6. To assess the linear range of the assay, HEK293/LRAT cells were transduced in triplicate with SPK-RPE65 at eight MOIs ranging from 3x103 – 2x106 vg/cell. Cell lysates were prepared from the 24 cell transductions and were analyzed in triplicate by incubation with all-trans-retinol and CRALBP. The amount of 11-cis-retinol produced was determined by LC-MS/MS. The plot of 11-cis-retinol as a function of MOI forms a sigmoidal curve, with a linear dose response between 3x104 and 5x105 MOI. Intra-assay reproducibility for the six MOIs above the limit of quantitation was good (CV=7–22%).

Figure 6. Dose dependency of 11-cis-retinol production

A

C B

*11-cis-retinol

Copyright © 2013 Elsevier. Cascella M., et al. Archives of Biochemistry and Biophysics. 2013. 539:187-195 .