Rhodopsin Genomic Loci DNA Nanoparticles Improve Expression and Rescue of Retinal Degeneration in a Model for Retinitis Pigmentosa

Please cite this article in press as: Zheng et al., Rhodopsin Genomic Loci DNA Nanoparticles Improve Expression and Rescue of Retinal Degeneration in a Model for Retinitis Pigmentosa, Molecular Therapy (2019), https://doi.org/10.1016/j.ymthe.2019.11.031

Original Article

Rhodopsin Genomic Loci DNA Nanoparticles Improve Expression and Rescue of Retinal Degeneration in a Model for Retinitis Pigmentosa Min Zheng,1 Rajendra N. Mitra,1 Ellen R. Weiss,2 and Zongchao Han1,3 1Department of Ophthalmology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; 2Department of Cell Biology and Physiology, The University

of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; 3Division of Pharmacoengineering & Molecular Pharmaceutics, Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA

The use of gene therapy may allow replacement of the defective gene. Minigenes, such as cDNAs, are often used. However, these may not express normal physiological genetic profiles due to lack of crucial endogenous regulatory elements. We constructed DNA nanoparticles (NPs) that contain either the mouse or human full-length rhodopsin genomic locus, including endogenous promoters, all introns, and flanking regulatory sequences of the 15–16 kb genomic rhodopsin DNA inserts. We transduced the NPs into primary retinal cell cultures from the rhodopsin knockout (RKO) mouse in vitro and into the RKO mouse in vivo and compared the effects on different functions to plasmid cDNA NP counterparts that were driven by ubiquitous promoters. Our results demonstrate that genomic DNA vectors resulted in long-term high levels of physiological transgene expression over a period of 5 months. In contrast, the cDNA counterparts exhibited low levels of expression with sensitivity to the endoplasmic reticulum (ER) stress mechanism using the same transgene copy number both in vitro and in vivo. This study demonstrates for the first time the transducing of the rhodopsin genomic locus using compacted DNA NPs.

INTRODUCTION The concept of gene therapy for delivering foreign genetic material into a patient’s cells was first raised in the 1960s.1 In 2017, for the first time, the US Food and Drug Administration (FDA) granted approval of a drug, LUXTURNA, to treat RPE65 mutations that cause retinitis pigmentosa (RP). Gene therapy is now emerging as one of the most important tools for human diseases. Most of the gene delivery systems utilize a synthetic cDNA of a reverse transcribed mRNA for the treatment. cDNAs are relatively short and can fit into viral vectors, such as adeno-associated virus (AAV). Indeed, gene therapy using cDNAs has been somewhat successful. However, these minigenes do not contain the gene’s regulatory sequences, such as the endogenous promoter/enhancer, introns, enhancers, and poly(A), which are important for determining when and how the gene is transcribed in vivo.2 Researchers have generated a number of transgenic animals using plasmid-based cDNA transgenic constructs. However, most of these transgenic animals do not accurately provide native gene functions.

Scientists began to realize that the problems resulted from the lack of endogenous regulatory elements necessary for physiological transgene expression in the cDNA constructs. These elements may not affect the rate of transcription; however, a growing body of evidence shows that they can significantly affect the stability of transgenes either at mRNA levels or protein levels.3,4. To overcome this issue, we generated earlier a nanoparticle (NP) to deliver an intron-containing short-form of a rhodopsin DNA for gene augmentation in a rhodopsin knockout (RKO) mouse model of retinitis pigmentosa (RP).5–7 Our results showed that this construct was expressed longer and resulted in greater phenotypic improvement than the cDNA nanoconstruct.5 Coinciding with the higher level of rhodopsin expression from the intron-containing rhodopsin DNA NP, we found that the rhodopsin cDNA NP, but not the intron-containing DNA NP, activates transgene silencing in the RKO mouse model.7 Furthermore, we demonstrated that a codonoptimized short genomic form of the rhodopsin nanoconstruct can significantly improve visual function in the P23H rhodopsin mutant mouse, which is a model for the dominant form of RP.6 These data indicate that non-coding DNA plays an important role in the regulation and control of gene expression following gene delivery. Here we further investigate the roles of the non-coding DNA in transgene expression and regulation of RNA production. We delivered the full-length human and mouse rhodopsin genomic DNAs, which contain the nucleotide sequences in their natural genomic context in vitro and in vivo. Polyethylene glycol-substituted polylysine (CK30PEG) diblock copolymer in combination with a cell-penetrating transactivator of transcription (TAT) (CK30PEG-TAT) was chosen as the delivery vector.6–14 In this work we report, for the first time, that our NPs are capable of delivering full-length human and mouse rhodopsin genes in

Received 17 July 2019; accepted 21 November 2019; https://doi.org/10.1016/j.ymthe.2019.11.031. Correspondence: Zongchao Han, MD, PhD, Department of Ophthalmology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA. E-mail: [email protected]

Molecular Therapy Vol. 28 No 2 February 2020

1

Please cite this article in press as: Zheng et al., Rhodopsin Genomic Loci DNA Nanoparticles Improve Expression and Rescue of Retinal Degeneration in a Model for Retinitis Pigmentosa, Molecular Therapy (2019), https://doi.org/10.1016/j.ymthe.2019.11.031

Molecular Therapy

RESULTS Human and Mouse Full-Length Rhodopsin Transgenes Expressed in Photoreceptor Primary Culture Cells

To confirm whether the full-length rhodopsin transgenes are expressed in photoreceptors, we used primary retinal cells at first passage as a model for transgene augmentation. Efficient transfection rates were obtained using primary retinal cells from 5- to 10-day-old RKO or age-matched wild-type (WT) mouse pups. As shown in Figure 1, expression of both rhodopsin and cone-specific S-opsin were detected in WT mice, but only S-opsin was identified in RKO mice. We also found both gDNA (gRho and gRHO) (Figure S1) and cDNA (cRho and cRHO) could be successfully transfected into primary retinal cells. Compared with WT cultures, the transfection efficiencies in primary retinal cells were approximately 30% of WT for gDNAs and 20% for cDNAs. The transfection efficiencies were confirmed by western blotting for rhodopsin (Figure 2A). We selected postnatal days 5–10 (P5–10) mouse retinas for primary cell cultures because studies showed that early retinal cells have higher survival rates and greater transgene expression.15,16 We and others have achieved transgene expression in RKO mice before rod photoreceptor degeneration using the cDNA or short forms of genomic rhodopsin genes;5–7 it is difficult to isolate primary retinal cells from adult mice. RKO photoreceptors rapidly degenerate from P15 even when the outer nuclear layer (ONL) is formed.17 Therefore, we delivered the vectors to primary cell cultures generated from RKO mice eyes prior to rod photoreceptor degeneration in this model.

Rhodopsin cDNA Transgenes, but Not gDNA Transgenes Provoked ER Stress in Photoreceptor Primary Cells Figure 1. Immunocytochemical Staining of Rhodopsin and S-opsin in Primary Retinal Cell Cultures Primary retinal cells from WT and RKO mice at postnatal day 5 (P5) were cultured. Linearized gRho or gRHO (without plasmid backbones) and their plasmid cDNA counterparts (cRho and cRHO) were transfected into primary retinal cell cultures from RKO mice. Primary retinal cells from age-matched WT mice were used as a control. At day 3 post-transfection, the cells were fixed with 4% paraformaldehyde (PFA) and stained for rhodopsin (1D4; green) and S-opsin (anti-S opsin; red) for cone cells. Expression of rhodopsin from the transgenes was evaluated with a Zeiss Axio fluorescence microscope. Scale bar, 100 mm.

primary retinal cell cultures and in RKO mice for transgene expression. These full-length gene constructs significantly delayed degeneration of the RKO mice for up to 5 months following a single subretinal administration. Compared to their cDNA counterparts, full-length genomic DNA particles show relatively greater physiological expression of rhodopsin, photoreceptor rescue, and lack of endoplasmic reticulum (ER) stress. Given that all non-coding and coding elements of the gene are included in the DNA NPs, our results have a significant impact in the field of gene therapy and demonstrate enhanced efficacy compared to the more commonly used cDNA delivery, which is important for improvements in precision medicine.

2

Molecular Therapy Vol. 28 No 2 February 2020

Rhodopsin is a multi-pass membrane protein that integrates into the disc membranes of rod photoreceptors in vivo. To investigate whether the transgenes were transported to the plasma membrane of primary retinal cells like WT rhodopsin, we performed transfection of RKO primary retinal cells with our rhodopsin expression cDNA plasmids and linear full-length gDNAs that lack a plasmid backbone. Agematched primary retinal cells from WT mice, in which there is no ER stress, were used as negative controls, and age-matched primary retinal cells from P23H mice, which are known to trigger ER stress, were used as positive controls.18,19 As shown by immunohistochemistry (IHC), both human and mouse gDNA-mediated rhodopsin protein expression predominantly localized to the cell membrane, whereas plasmid cDNA-mediated rhodopsin expression was detected in both the cytoplasm and the cell membrane at 3-day post-transfection (Figure 2B), indicating that rhodopsin from cDNA transgenes may be partially (or completely) trapped in the ER compared to rhodopsin expressed from gDNA. We then asked whether the accumulation of the rhodopsin protein in the cytoplasm will trigger photoreceptor death. We performed TUNEL staining and found no significant increase in the number of apoptotic cells following transfection, indicating that although the plasmid cDNA transgenes were under pressure of ER stress, the stress was not severe enough to cause extensive cell death (Figure S2).

Please cite this article in press as: Zheng et al., Rhodopsin Genomic Loci DNA Nanoparticles Improve Expression and Rescue of Retinal Degeneration in a Model for Retinitis Pigmentosa, Molecular Therapy (2019), https://doi.org/10.1016/j.ymthe.2019.11.031

www.moleculartherapy.org

Figure 2. Human and Mouse Rhodopsin Expressed from Transgenes in Primary Photoreceptor Cell Cultures from P5 RKO Mice Retinas from P5 RKO mice were processed for primary cell culture and were transfected with a human linear full-length rhodopsin DNA (gRHO), a human cDNA rhodopsin plasmid (pCAG-cRHO), a mouse a full-length linear rhodopsin DNA (gRho), or a mouse cDNA rhodopsin plasmid (pCAG-cRho) via the Nucleofector 2b Device. (A) Western blotting of proteins from day 3 post-transfection with 1D4 (1:500, for human and mouse rhodopsin) and anti-human rhodopsin (1:500, for human rhodopsin only) antibodies for western blot analyses. b-actin served as an internal control. Age-matched WT mouse primary photoreceptor cells were used as a positive control. (B) gDNA, but not cDNA, transfection promoted rhodopsin trafficking to the cell membrane in primary retinal cell cultures from P5 RKO mice. On day 3 post-transfection, cells were immunostained with 1D4 (1:500, for human and mouse rhodopsin, green) and anti S-opsin (1:500, for S opsin cone cell, red) antibodies, and DAPI counterstaining of nuclei (blue). Age-matched WT and P23H rhodopsin primary retinal cells were used as controls. Scale bar, 4 mm.

Dose-Response Functional Rhodopsin Gene Expression In Vivo

Having determined the relative expression efficiencies of different transgenes in primary retinal cells, we sought to confirm our observations in vivo in the RKO model. Previously, we achieved an optimal dose of 0.3 mL (for newborn pups, 1.29 mg/eye) and 1 mL (for adult mice at a concentration of 4.3 mg/mL) by delivering these reagents as plasmid-based compacted DNA into the subretinal space. At those doses, high transgene levels were achieved in photoreceptors2 and retinal pigment epithelial cells.4 In the present experiment, we used the entire genomic locus of rhodopsin as the transgene. Multiple doses of 0.15, 0.3, 0.6, 0.9, 1.2, and 1.5 mg/eye of NP-gRho diluted in 0.3 mL saline were delivered by subretinal injection in P1 RKO mice. We chose this range based on our previous studies,5–7 which we believe will cover the expected maximum tolerated dosage. We determined mRNA expression levels of rhodopsin by using qRTPCR at 60 days post-injection (PI-60 days) (Figure 3). The predicted size of the PCR product and the sequences of the specific primers are listed in Table 1. Our results showed that CK30PEG-TAT gDNA NPs can carry a fulllength genomic form of rhodopsin genes (gRho or gRHO) with all endogenous regulatory elements including an endogenous promoter, enhancers, suppressors, and introns for gene replacement therapy in RKO mice. Our first generation of CK30PEG with a short form of rhodopsin transgene when injected in RKO mice resulted in
10% rhodopsin transgene expression compared to WT mouse rhodopsin expression.5,7 Our 2nd generation of CK30PEG-TAT can carry the full-length of rhodopsin transgene and achieved
50% of transgene expression with doses between 0.9 and 1.2 mg. Therefore, CK30PEG-TAT NP can achieve superior rhodopsin transgene expression with a relatively low dose of DNA (1.2 mg/eye). In addition, rhodopsin mRNA expression increased in a dose-dependent manner from 0.6 to 1.2 mg. A higher dose did not increase the

efficiency of transfection (Figure 3). As a result, for the remainder of this study, we have used equal DNA copy number of 1.2 mg/eye of gDNA NPs and 0.6 mg/eye cDNA NPs for subretinal injection (1.2 mg/eye of gDNA at
15,000 bp, which corresponds to 0.6 mg/mL of plasmid cDNA at 7,500 bp in DNA copy numbers; http://cels.uri.edu/gsc/cndna.html).

Full-Length Human and Mouse Rhodopsin Transgenes Expressed in RKO Mice over an Extended (up to 5 Months) Period of Time

To investigate whether the strategy is effective and can achieve sustained transgene expression, we injected equal copy numbers of NP-gRho, -cRho, -gRHO, and -cRHO subretinally into P1 RKO eyes. Un-injected or saline-injected RKO and WT mouse retinas were used as controls. Retinas were collected at PI-1 month, PI-3 months, and PI-5 months and analyzed for transgene expression. Both human and mouse rhodopsin mRNA from gDNA NPs were expressed for 5 months in RKO mice at
50% of WT rhodopsin. While we found there was slightly higher transgene expression in the gRho DNA NP- than in gRHO DNA NP-injected eyes, no significant differences were observed in the rhodopsin mRNA levels between human and mouse gDNA injected eyes from PI-1 month to PI5 months (Figure 4A). In contrast, injection of human and mouse cDNA resulted in a lower expression of rhodopsin mRNA (from
15% of WT rhodopsin at PI-1 month to
0%–1% of the WT rhodopsin at PI-3 months). At PI-5 months, no rhodopsin expression was detected in cDNA NP-treated mice. Compared to their gDNA counterparts, significant differences were observed at each time point (Figure 4A). The rhodopsin protein expression pattern for the transgene resembled the gene transcription patterns. Expression of the rhodopsin protein from the full-length gDNA ranged from 50% to 40% of WT expression levels from PI-1 to PI-5 months, whereas

Molecular Therapy Vol. 28 No 2 February 2020

3

Please cite this article in press as: Zheng et al., Rhodopsin Genomic Loci DNA Nanoparticles Improve Expression and Rescue of Retinal Degeneration in a Model for Retinitis Pigmentosa, Molecular Therapy (2019), https://doi.org/10.1016/j.ymthe.2019.11.031

Molecular Therapy

present in the ONL at the central retina. However, gDNA-treated mice had approximately 50% of the thickness of WT outer segments and 3–7 rows of nuclei in the ONL (photoreceptor nuclei) compared to 11–13 rows in the ONL and maximal thickness of outer segments in WT retinas. The cDNA-treated RKO mice exhibited 0–1 rows of nuclei in the ONL and no outer segments, indicating a nearly complete lack of rhodopsin and severe degeneration of the photoreceptors. The number of rows was significantly increased (***p < 0.001) for both gDNA-treated compared to both cDNA-treated groups (Figure 5C).

Figure 3. Dose Response of Rhodopsin Transgene Levels in RKO Mice following Subretinal Injection Multiple doses of gRho and gRHO NPs were diluted in saline and delivered via subretinal injection to postnatal day 3 (P3) RKO mice (0.3 mL/eye). At 2 months postinjection (PI-2 months), the retinas were collected and levels of rhodopsin mRNA expression were examined by qRT-PCR. The rhodopsin expression increased in a dose-dependent manner at a range of 0.5–1.5 mg; at a dose of 1 mg, the transgene level was approximately 50% of endogenous rhodopsin mRNA expression and then plateaued at higher doses. Data are mean ± SEM, n = 4–6 eyes/group.

expression of rhodopsin from the cDNA showed less rhodopsin expression: from 10% at PI-1 month, 0%–1% at PI-3 months, and totally undetectable at PI-5 months (Figure 4B). Significant differences were observed (p < 0.001) comparing both gDNA-treated to the cDNA-treated groups at each time point (Figure 4C). The same membrane was immunoblotted with anti-IRE1a (inositol-requiring enzyme 1 a) to determine whether ER stress occurs during the treatment. We found that the level of IRE1a was significantly greater in cDNA NP-injected RKO mice than in gDNA NP-injected RKO mouse retinas at PI-1 month and -3 months (Figures 4B and 4D). Furthermore, it is interesting to note that although the rhodopsin mRNA transcribed from cDNA transgenes was undetectable at PI-5 months, we found that the vector genomes were still present and the amount of the vector genomes was comparable to that present at PI-1 months in the retina, suggesting that the cDNA forms of the transgenes were fully silenced through epigenetic control mechanisms at PI-5 months. The persistence of vector genome until 5 months was confirmed using PCR with primers for a specific region in exons 1 and 2 of WT mice but mismatched with RKO mice. (Figure 4E). Additionally, human and mouse rhodopsin transgenes were identified by PCR using human-specific, mouse specific, and humanmouse specific primers to rhodopsin (Figure S3). Human and Mouse gDNAs Structurally and Functionally Rescue the Photoreceptors of RKO Mice

To evaluate whether the transgenes can rescue structural changes resulting from the loss of rhodopsin in RKO mice,17 we stained retinal sections of RKO mice at PI-5 months with H&E (Figures 5A and 5B). At this age, photoreceptors in untreated RKO retinas had no outer or inner segments and less than 1 row of photoreceptor nuclei was

4

Molecular Therapy Vol. 28 No 2 February 2020

Next, we sought to evaluate whether our NPs could functionally rescue the RKO degeneration phenotype. Full-field electroretinography (ERG) was performed at PI-1 month, -3 months, and -5 months (Figures 6A– 6C). At PI-1 month, a 50% functional rescue of the rod response after gDNA NPs delivery was observed using a 0.01 cd s/m2 flash and measuring the b-wave amplitudes, whereas only a 10% rod functional rescue was observed after injection of cDNA NPs. By PI-3 months, the amplitudes of the rod response following a 0.01 cd s/m2 flash in gDNA-injected retinas were reduced to 40% of WT, whereas in cDNA-treated mice, the rod response was nearly undetectable. At PI-5 months, the rod scotopic ERG response was about 35% of WT in gDNA treated mice; however, we could not detect any ERG response in cDNA-treated mice, indicating only gDNA treatment rescued rod photoreceptors in RKO mice (Figure 6A). Separate comparisons were made at each time point. Significant differences were observed comparing gDNA-treated with cDNA-treated mice at each time point (*p < 0.05, **p < 0.01, and ***p < 0.001) (Figure 6D). Similar results were observed with the rod-cone mixed scotopic a- and b-wave responses at 2.25 cd s/m2, which were comparable to that of rod response in cDNA- and gDNA-treated mice, respectively (Figures 6B and 6D). Furthermore, we found that gDNA treatment inhibited progressive degeneration of cone cells in RKO mice that is caused by rod system dysfunction. In contrast, the cDNA-treated RKO mice had progressive degeneration of their cone cells based on reduced photopic b-wave amplitudes of the cone-mediated response after a 30 cd s/m2 flash (Figure 6C). The b-wave in these mice was 70% of WT at PI-1 month, then decreased significantly to 30% of WT at PI-3 months, and was undetectable at PI-5 months (Figure 6D). In contrast, in gDNA-treated RKO mice, the b-wave was maintained at relatively high levels: 90% of WT at PI-1 month, then dropped slightly to 80% of WT at PI-3 months, and to 70%–80% of WT at PI-5 months. Notably, there was no significant difference between each time point in gDNA-treated mice (Figure 6D), indicating functional stability of the retina over a 5-month period. cDNA NPs Expression Led ER Stress Response in RKO Mice

Improper transgene expression may cause a wound response in the host cell.20 Incorrect folding of the transgene-expressed protein may trigger ER stress, which may result in ER-associated protein degradation, affecting the efficacy of transgene protein expression. To determine whether our vectors increased ER stress, we assessed chaperone binding immunoglobluin protein (BiP/GRP78), IRE1a,

Please cite this article in press as: Zheng et al., Rhodopsin Genomic Loci DNA Nanoparticles Improve Expression and Rescue of Retinal Degeneration in a Model for Retinitis Pigmentosa, Molecular Therapy (2019), https://doi.org/10.1016/j.ymthe.2019.11.031

www.moleculartherapy.org

Table 1. List of Primer Sets Used for PCR and RT-PCR in This Study Primer Name

Forward Primer (50 - > 30 )

Product Size (bp)

5 -AGCACCCTCTACACCTCTCTG-3

50 -AACGCCCATGATGGCATGG-30

218

Mus rhodopsin

50 -CACCACCCTCTACACATCACTC-30

50 -CACACCCATGATAGCGTGA-30

212

Hu/mus rhodopsin

50 -CTTCCCCATCAACTTCCTCA-30

50 -CGGAAGTTGCTCATGGGCTT-30

Mus BiP

50 -CTTTGATCAGCGGGTCATGG-30

50 -AGCTCTTCAAATTTGGCCCG-30

219

Mus IRE1a

50 -CAATCGTACGGCAGTTGGAG-30

50 -CTCCCGGTAGTGGTGTTTCT-30

170

Mus CHOP

50 -ACGGAAACAGAGTGGTCAGT-30

50 -AGACAGACAGGAGGTGATGC-30

0

5 -TGTTACCAACTGGGACGACA-3

0

Reverse Primer (50 - > 30 )

Hu rhodopsin

b-actin for hu/mus

0

0

0

5 -GGGGTGTTGAAGGTCTCAAA-3

0

287 (mus) 287 (hu)

226 165 385 (mus cDNA)

Hu/mus rhodopsin (for vector genome)

50 -GTGGTACGCAGCCCCTTCGA-30

50 -CGGAAGTTGCTCATGGGCTT-30

385 (hu cDNA) 1,774 (mus gDNA) 2,069 (hu gDNA)

and C/EBP homologous protein (CHOP) mRNA levels by RT-PCR at PI-1 month, -3 months, and -5 months. As we predicted, both BiP/ GRP78 and IRE1a mRNA levels were significantly greater in cDNA NP-injected RKO mice than in gDNA NP-injected RKO mouse retinas at PI-1 month and -3 months, suggesting that cDNA NP delivery induced ER stress (Figure 7; see also Figure 4D). Failure to detect ER changes after PI-3 months indicates that the cDNA transgenes are fully silenced. Interestingly, other than a positive control (HEK293 cells treated with tunicamycin), we did not observe elevated CHOP mRNA or protein expression under these conditions (data not shown). CHOP is generally considered an indicator of more severe ER stress that initiates programmed cell death in a later phase of the ER stress response,21–23 suggesting that the level of ER stress in these mice may not be sufficient to cause cell death.

DISCUSSION The idea of using intact genomic loci for gene targeting has developed over the past few decades. Studies have used adenovirus,24 herpes simplex virus (HSV-1),25,26 and artificial chromosomal (AC) vectors,27–31 such as yeast AC (YAC), bacterial AC (BAC), and human AC (HAC) for gene targeting. Although the results have shown very accurate and physiological long-term transgene expression in vitro and in vivo, a number of challenges are associated with the use of these viral vectors and ACs, e.g., insertional mutagenesis, immune responses, and complex mechanisms of the vector design and construction, as well as problems with the safety and efficiency of delivery procedures, which have yet to be resolved in clinical practice.32–35 We and others using NP gene therapy strategies have shown long-term expression from transgenes in the retina and improved phenotypic and functional rescue in RP mouse models.5,10,11,36–38 These particles are unique and offer a number of advantages over viral or artificial chromosomal vectors. One of the biggest advantages is that NPs can carry larger DNA sequences through simple electronic charge machinery.9,39 There is no limitation on the size of the gene to be delivered,10,40 and therefore genes can be delivered that contain

endogenous regulatory elements. By adding TAT to the nanoparticle, additional membrane-penetrating ability is provided for delivery of genes into the cells.6,14 In this study, we provide evidence and feasibility of transferring the full-length genomic DNA of the rhodopsin gene, which contains all the regulatory sequences necessary for expressing the fully functional protein. We found that this system is critical for the development of a stable and regulated gene expression system at physiological levels for targeted ocular gene therapy. Our results are supported by our and others’ previous studies demonstrating the potential of an intact genomic locus delivery approach for structural and functional rescue of a genetic defect.5–7,25,26,41 We demonstrate that NPs have the ability to transfer larger genes, which can overcome limitations of traditional gene therapy vehicles. Our study provides insight into a new concept of gene therapy associated with using a full-length transgene and could be a promising tool for future applications of gene therapy. Historically, rhodopsin transgenes have been primarily studied in numerous stable cell lines and genetic animal models. However, studies on primary retinal cells have been limited due to several technical challenges. Primary cells are the most biologically and physiologically relevant model system for testing therapeutic strategies. Previously, primary retinal cell cultures have been reported to have different cellular expression profiles compared to the retina in vivo.16,42–44 Co-culture of retinal cells with several critical extracellular matrix elements has been shown to support photoreceptor survival and proper signaling due to providing a more natural environment.45,46 The method we used here results in survival of more than 60% of the photoreceptors without losing their identities (i.e., rhodopsin and S-opsin expression) for at least 7 days. Studies have shown that non-coding DNAs not only play an important role in gene regulation but also a vital role in ER function. Several transcription factors and cofactors have been confirmed to be connected to

Molecular Therapy Vol. 28 No 2 February 2020

5

Please cite this article in press as: Zheng et al., Rhodopsin Genomic Loci DNA Nanoparticles Improve Expression and Rescue of Retinal Degeneration in a Model for Retinitis Pigmentosa, Molecular Therapy (2019), https://doi.org/10.1016/j.ymthe.2019.11.031

Molecular Therapy

Figure 4. Human and Mouse Rhodopsin Expressed from gDNA and cDNA Transgenes in RKO Mice but Showed Different Levels and Period of Time of Expression (A) RT-PCR was performed with primers specific for both mouse and human rhodopsin. Real-time PCR results are shown for the expression levels of mRNA from gDNA NPsand cDNA NPs-treated and un-treated RKO mice retinas (n = 4–6). Mean rhodopsin mRNA expression levels were normalized to b-actin (internal reference) and expressed as a percentage of that in age-matched WT. (B–D) Western blots for human and mouse rhodopsin (1D4, binds to the rhodopsin C-terminal 9 amino acids, TETSQVAPA), human rhodopsin only (Hu-RHO, binds at the human rhodopsin C terminus at amino acids 310–339), IRE1a, and b-actin antibodies were used to assess IRE1a and protein expression in the retina at PI-1, -3, and -5 months. Un-injected RKO and WT mice were used as controls. Protein levels were normalized to b-actin and expressed as a percentage of protein measured in age-matched WT mice. Data measuring levels of rhodopsin mRNA and protein in gDNAtreated RKO mice compared to cDNA-treated RKO mice represent mean ± SEM, n = 3–5 eyes/group. *p < 0.05, **p < 0.01, and ***p < 0.001 by two-way ANOVA with Bonferroni’s post hoc tests. (E) To evaluate the persistence of the vector genome, we performed PCR using primers designed on the exons 1 and 2 but mismatched with RKO mice at PI-3 and -5 months.

the unfolded protein response (UPR) pathway.47,48 Some cis-acting elements have also been found to directly or indirectly affect ER quality control machinery.47,49,50 Instability of mRNAs is linked with ER stress.51,52 All these studies suggest that physiological gene expression and regulation have influenced the regulation of the UPR. ER stress is known to be caused by retinal degeneration due to various factors, including genetic mutations, light damage, and pharmacological inhibitors.21,53–58 Rhodopsin is an integral membrane protein that is synthesized and folded in the ER. Studies have shown that defective rhodopsin (e.g., unfolded or improperly folded protein) is unable to pass through the ER quality control point to translocate to the plasma membrane in cultured cells or to the outer segments in vivo but rather is retained in the ER.54,59 To determine the localization of the rhodopsin expressed from transgenes, we used confocal microscopy to investigate the rhodopsin protein trafficking behaviors in primary retinal cell cultures transfected with gDNA or cDNA transgenes. We found that rhodopsin generated from gDNA successfully translocated to the plasma membrane, which was similar to the translocation pattern of rhodopsin in WT controls, whereas rhodopsin in cDNA-transfected cells was found scattered in both the cytosol and cell membrane, indicating that protein from the cDNA transgenes might be partially trapped in the ER (Figure 2). These data suggest that non-coding regions included in the gDNA for rhodopsin are important for its proper translocation. We found no difference in the numbers of apoptotic cells between the cDNA and gDNA transfections, indicating that ER stress caused by misfolded rhodopsin expressed from cDNAs is an early event and

6

Molecular Therapy Vol. 28 No 2 February 2020

does not reach the level that leads to appreciable photoreceptor death. Our earlier studies showed that delivery of cDNA to RKO mouse eyes resulted in partial epigenetic transgene silencing compared to that of the short genomic transgene counterparts.5,7 This is important because it suggests that use of gDNA transgenes may lead to physiological/ natural protein expression patterns and levels. Although this hypothesis needs to be tested further, it is clear that rhodopsin mRNA from a cDNA transgene is not expressed at the level observed from the endogenous gene. Therefore, the expression level (e.g., stability of expression from a cDNA transgene) might be compromised by some factor. The stability of the mRNA from the transgene could be short-lived or permanently suppressed by the host epigenetic system. To further investigate the stability and long-term expression from the transgene, we performed in-depth in vivo studies and quantitative comparisons of gDNAs and cDNAs in RKO mice. DNAs were compacted into CK30PEG-TAT NPs. CK30PEG has been widely utilized for its many advantages, including its safety, large capacity, and ability to drive long-term gene expression in both dividing and non-dividing cells.7–13 Our results demonstrated that gDNA achieves long-term stable expression of rhodopsin that is nearly 50% of endogenous levels in WT mice throughout the 5-month test period, whereas the cDNA constructs of the same copy number only achieved
5% of WT rhodopsin expression within the first month and rhodopsin could not be detected after PI-3 months. Our results indicate that the gDNA construct CK30PEG-TAT particles are more efficient and more stable than their

Please cite this article in press as: Zheng et al., Rhodopsin Genomic Loci DNA Nanoparticles Improve Expression and Rescue of Retinal Degeneration in a Model for Retinitis Pigmentosa, Molecular Therapy (2019), https://doi.org/10.1016/j.ymthe.2019.11.031

www.moleculartherapy.org

Figure 5. gDNA NPs Delivery Partially Rescued Retinal Structure in RKO Mice after 5 Months of Treatment (A) Inserts (from B) show images of retinal structure after a single subretinal injection in RKO mice at PI-5 months. N, nasal; T, temporal. Scale bar, 20 mm. (B) Representative histology of H&E stained retinal cross-sections from gDNA- and cDNA-treated RKO mice at PI-5 months. WT and saline-injected RKO were used as controls. Scale bar, 200 mm. (C) Quantification of the number of rows in the ONL. Data measuring the number of rows in the ONL in gDNA-treated RKO mice compared to cDNA-treated RKO mice represent mean ± SEM, n = 3–5, *p < 0.05, **p < 0.01. ONH, optic nerve head; ONL, outer nuclear layer; INL, inner nuclear layer; IS, inner segment; OS, outer segment.

cDNA counterparts, which supports our hypothesis that the rhodopsin transgene produced by gDNA is able to more fully recapitulate the expression pattern of the endogenous gene. Although the underlying molecular mechanisms require further investigation, the genomic locus contains the most native elements for regulation of gene expression and therefore can achieve high levels of post-translational processing and functional activity. Long-term (up to PI-5 months) transgene expression in vivo may also be due to the scaffold/matrix attachment region (S/MAR), cis-regulatory elements of the gDNA, which enable extended stability of the transgene, given the non-dividing nature of the photoreceptors. We are making progress in defining the non-coding elements that are responsible for this behavior. Interestingly, the vector genomes of the cDNA NPs were still detectable at PI-5 months and were comparable to that of their gDNA NPs at PI-1 month but expression from the transgenes was completely undetectable, indicating that

cDNA NP transgenes underwent silencing. This is consistent with data from several other laboratories demonstrating that minigene constructs without the necessary non-coding regions generated only low levels or inactive protein expression.60,61 Likewise, the human gene is commonly used in mouse models to study human diseases. However, the human gene has a greater number of cis-acting elements than the mouse gene. There are 18 amino acid differences between human and mouse rhodopsin cDNA.62 To date, there have been no studies showing variability between gRHO and gRho expression. Although the human rhodopsin gene has been reported to successfully express rhodopsin and rescue degeneration in RKO mice,62 we speculate that differences between RHO and Rho exist that may cause differential transcription and post-translational modifications. A substantial amount of research is needed to examine these speculations. Our current study shows that gRho delivery exhibits slightly higher rhodopsin

Molecular Therapy Vol. 28 No 2 February 2020

7

Please cite this article in press as: Zheng et al., Rhodopsin Genomic Loci DNA Nanoparticles Improve Expression and Rescue of Retinal Degeneration in a Model for Retinitis Pigmentosa, Molecular Therapy (2019), https://doi.org/10.1016/j.ymthe.2019.11.031

Molecular Therapy

Figure 6. gDNA NPs Delivery Partially Rescued Retinal Electroretinography Responses in RKO Mice after 5 Months of Treatment Full field electroretinography (ERG) for scotopic and photopic responses in dark-adapted RKO mice were measured at PI-1, -3, and -5 months as described in the Materials and Methods. (A) Representative rod responses at 0.01 cd s/m2. (B) Mixed rod and cone responses at 2.25 cd s/m2. (C) Cone responses at 30 cd s/m2. (D) Quantification of the average amplitudes of scotopic a- and b-waves and the photopic b-wave. Age-matched WT and saline-injected RKO mice were use as controls. Data measuring ERG responses in gDNA-treated RKO mice compared to cDNA-treated RKO mice represent mean ± SEM, n = 6–10, *p < 0.05, **p < 0.01.

mRNA and protein expression, as well as functional stability (Figures 4, 5, and 6), than that of gRHO. Whether or not the gRHO will generate exactly the same pattern of transgene expression as gRho in RKO mice is worthy of further study.

Therefore, this work is the first demonstration of successful delivery of the native genomic rhodopsin sequence and its critical importance for higher levels of functional and structural rescue in a murine model of RP.

Previously, we compared the intron-containing short form of the genomic rhodopsin DNA NPs and intron-lacking cDNA rhodopsin NPs in RP mice. We found that exon-containing DNA NPs were not only able to enhance expression from transgenes but also tended to have fewer epigenetic modifications than their cDNA counterpart.5,7 To investigate whether the transgenes have any effect on ER stress in vivo, we measured mRNA and protein levels of ER stress markers, including IRE1a, BiP, and CHOP. We found that expression of IRE1a and BiP were significantly higher in cDNA-treated RKO retinas than in RKO retinas containing gDNA, suggesting that cDNA delivery might have triggered ER stress, which further highlights the importance of a complete genomic locus for physiological gene expression.

In summary, we have used the genomic rhodopsin DNA locus as a transgene for gene augmentation. Our results suggest that this is a highly significant improvement compared to the cDNA construct. The combined use of the nanoparticle delivery system with gDNAbased gene therapy has great potential to overcome many problems caused by the use of viral or non-viral plasmid-based gene transfer and represents a big advantage over the traditional cDNA-based therapy. We take advantage of the high capacity of our NPs and their ability to deliver and sustain transgene expression in the absence of vector integration. Our data indicate that delivery of the entire genomic transgene locus in the absence of a bacterial backbone could potentially bypass ER quality control mechanisms for transgene expression. We demonstrate for the first time long-term gene rescue using the full-length genomic rhodopsin locus in the RKO mouse model of RP disease. Our work offers several significant advantages over viral (e.g., adenovirus and HSV viral vectors) and artificial chromosome vectors in gene therapy in terms of bio-safety, large capacity, expression equivalent to the native gene, and a straightforward

The same mouse and human full-length functional genomic rhodopsin DNA loci that we utilized in this study have previously been used in a transgenic animal63 and in gene structural studies,64 but have never been investigated for efficacy as a tool for gene therapy.

8

Molecular Therapy Vol. 28 No 2 February 2020

Please cite this article in press as: Zheng et al., Rhodopsin Genomic Loci DNA Nanoparticles Improve Expression and Rescue of Retinal Degeneration in a Model for Retinitis Pigmentosa, Molecular Therapy (2019), https://doi.org/10.1016/j.ymthe.2019.11.031

www.moleculartherapy.org

Figure 7. cDNA NPs Delivery Resulted in ER Stress Response in RKO Mice Retinal lysates from gDNA NPs- and cDNA NPs-treated mice were subjected to RT-PCR to detect the expression of IRE1a and BiP. b-actin was used as an internal control. Data are means ± SEM (n = 3–5).

mechanism, making it an attractive and potentially more powerful genetic tool to be exploited for precision medicine approaches to RP caused by rhodopsin mutations. Beyond this, we also highlight that this novel platform could be adapted to treat many other genetic disorders, for which gene replacement therapy could be a treatment.

removed the 15-kb gRho and 16-kb gRHO genomic DNAs from their EMBL3 phage vectors and cloned them into a Gibson Assembly-ready vector, pUCGA (2,752 bp), named pUC-gRho and pUC-gRHO, respectively. Correct vector identity was verified by restriction enzyme digestion and sequence analysis. GA L-arm EMBL3 Forward

MATERIALS AND METHODS Animals

The RKO mice were provided by Dr. Janis Lem (Tufts Medical Center, Boston, MA, USA) under a material transfer agreement. Age-matched WT (C57BL/6J, Jackson Laboratory) mice were used as controls. All experimental animals were managed in accordance with the principles approved by the University of North Carolina at Chapel Hill Institutional Animal Care and Use Committee and were handled following the Guide for the Care and Use of Laboratory Animals and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Research. Vectors and Constructs

Mouse and human rhodopsin cDNAs from pEPI-cRho5 and pRK5cRHO (kindly provided by Dr. Jeremy Nathans, Johns Hopkins University School of Medicine) were subcloned into a pCAG plasmid (without GFP) driven by the CAG promoter (the cytomegalovirus enhancer fused to the chicken beta-actin promoter) by standard cloning. The full-length 16-kb human gRHO gene was kindly provided by Dr. Thaddeus Dryja (Novartis Institutes and Harvard Medical School). The full-length 15-kb mouse gRho gene was kindly provided by Dr. Al-Ubaidi (University of Houston). These gRHO or gRho genes contain their native, endogenous opsin promoters and poly(A) signals.63,64 These DNAs were originally cloned into a bacteriophage vector. For quick and easy amplification of these large genomic DNAs, we sub-cloned the gRho and gRHO genes into a pUCGA 1.0 plasmid (Synthetic Genomics, Cat. No. GA1020, modified from pUC19) though Gibson Assembly (see below and Figure S1). Cloning Full-Length Mouse and Human Rhodopsin into the pUCGA Vector

The Gibson Assembly 2-step method (Gibson Assembly Ultra Kit, Cat No. GA1200) and Lucigen electrocompetent cells (Lucigen) were used to carry out the assembly process. By adding sequence that overlaps (blue letters; see below) the pUCGA 1.0 via PCR (PrimeSTAR GXL DNA Polymerase, Takara), we successfully

50 -GAGCACATCTCGTTCGCTATTCAGGGATTGGATGCCATGGTGTCCGACTTATGCCCGAGAAGATGTTGAG-30

GA R-arm EMBL3 Reverse

50 -CATCCACGCCTGAGATCTAGACAAACTTCCAAATGCAAGAGCAAAGACGAAAACATGCCACACATGAGGA-30

DNA NP Compaction

DNA NPs were compacted as previously described.5–7,39,65,66 Briefly, the TAT peptide was linked to CK30PEG by standard bioconjugation.6 CK30PEG-TAT was further lyophilized and confirmed by 1H-nuclear magnetic resonance (NMR; Varian 400MR) spectroscopy and Fourier transform infrared spectroscopy (FTIR, Nicolet 380 FTIR, Thermo Fisher Scientific) to ensure the authenticity and purity for subsequent in vivo studies. The full-length linearized 15-kb gRho and 16-kb gRHO genes, which contain a 6- or 4.8-kb sequence upstream of the translation initiation codon and a 3.5- or 6.2-kb sequence downstream of the terminal codon of the 30 untranslated region of mouse or human rhodopsin, respectively,63,64 were removed from their pUC vectors using the restriction enzyme, SalI. The DNA fragments were extracted using agarose gel purification and dissolved in endotoxin-free water. Then, the two purified genomic DNAs (gRho and gRHO) along with mouse and human rhodopsin cDNA plasmids (pCAG-cRho and pCAG-cRHO, respectively) were separately mixed with CK30PEG-TAT following an earlier protocol.5–7,39,65,66 The final DNA NPs were solubilized in saline (0.9% sodium chloride) and concentrated to 2.5 mg DNA/mL. The successful compaction of the DNA NPs was confirmed by an agarose gel retardation assay, trypsin digestion assay, and DNase I digestion assay as previously described.6,7 Retinal Primary Cell Culture

The retinas from P5–10 RKO or WT mice were used for primary culture. We followed procedures described previously44 with some

Molecular Therapy Vol. 28 No 2 February 2020

9

Please cite this article in press as: Zheng et al., Rhodopsin Genomic Loci DNA Nanoparticles Improve Expression and Rescue of Retinal Degeneration in a Model for Retinitis Pigmentosa, Molecular Therapy (2019), https://doi.org/10.1016/j.ymthe.2019.11.031

Molecular Therapy

modifications by including RPE cells. Briefly, retinas were isolated and subsequently rinsed 2–3 times with Ringer’s solution. They were then cut into
2 mm2 pieces and incubated with 2 units of pre-warmed papain in 25 mL of activator solution (1.1 mM EDTA, 5.5 mM L-cysteine, 60 mM b-mercaptoethanol) in a 37 C cell culture incubator for 30 min. The reaction was stopped by adding 1 mL of 10% fetal calf serum (FCS) in DMEM along with 25 U of DNase I to digest dead cells. The tissue was gently homogenized to make single-cell suspensions using a 1 mL pipette and centrifuged at 50 g for 5 min. The cell pellet was rinsed with DMEM and centrifuged 1–2 times before re-suspension in DMEM containing brain-derived neurotrophic factor (BDNF, 50 ng/mL), ciliary neurotrophic factor (CNTF, 10 ng/mL), murine fibroblast growth factor (FGF-2, 30 ng/mL), and 10% FCS. At this point, the retinal primary cells were ready for counting and direct DNA transfection with Nucleofector 2b (Lonza).

were performed at P1 via a transvitreal approach under a surgical microscope as previously described.7,10 Briefly, P1 mice were anesthetized by placement in crushed ice for 1–3 min. The eyelid was cut, and the cornea was punctured using a 30G BD insulin syringe needle. A 35G blunt-end needle attached to a 10 mL Nanofil syringe (World Precision Instruments, Sarasota, FL, USA) controlled by a Micro4 Microsyringe injection system (World Precision Instruments, Sarasota, FL, USA) was inserted into the puncture site. NPs (0.1–0.5 mL, 2.5 mg/mL) in saline were delivered into the subretinal space. Saline injected of the same volume (vehicle) were used as controls. After injection, the needle was left in place for a few seconds to allow full treatment delivery before being slowly withdrawn. The eyelid was returned to its original position and a drop of Triple Antibiotic (Equate, WalMart, Bentonville, AR, USA) ointment was applied. Mice were then placed on a warm pad at 37 C until fully awake prior to placing them with the dam.

Nucleofection and In Vitro Immunocytochemistry

Histological Analysis

Equal DNA copy numbers (see Results section) of 1.2 mg linear gRho or gRHO and 0.6 mg of pCAG-cRho or pCAG-cRHO were added to 106 primary retinal cells in 100 mL Nucleofector Solution (A-33, the installed program). After transfection, the cells were plated with 500 mL pre-warmed DMEM medium containing 10% FCS, BDNF, CNTF, and FGF-2 in a 6-well cell culture plate and incubated at 37 C in 5% CO2 for 3–6 h. An additional 1.5 mL of culture medium was added and the cells were cultured for 48 h as described above. We also cultured the cells directly on cell culture slides for confocal images. More than 60% of these cultured cells were photoreceptors. In the photoreceptor population, approximately 85% were rods and 15% were cones, which can survive for at least 7 days without losing their individual characteristics (i.e., rhodopsin and cone opsin expression) and functions in vitro under these conditions (Figure 1).

At PI-5 months, photoreceptor structure and retinal morphology were evaluated by histological analysis (UNC Histology Research Core Facility). Mouse eyes were collected, fixed in cold methanol/acetic acid solution, and embedded in paraffin as described elsewhere.67,68 Sections of 5 mm thickness were cut from the central (optic nerve) block and stained with H&E for structural integrity, which was determined by light microscopy.

3 days after transfection, retinal primary cells from WT and RKO mice were fixed with 4% PFA for 30 min and washed briefly in PBS. Then, the slides containing cells were blocked for 60 min at room temperature (RT) with antibody dilution buffer (5% donkey serum, 3% BSA, 0.05% Triton X-100 in PBS) to reduce nonspecific binding. The mouse monoclonal rhodopsin antibody, 1D4, (1:1,000, Santa Cruz Biotechnology, Dallas, TX) was used to stain for rhodopsin and a rabbit polyclonal anti-S-opsin antibody (1:1,000, Novus Biologicals) was used to stain cone cells. After rinsing with PBS, the slides were incubated for 1 h at RT with donkey anti-mouse Alexa Fluor 488 (Molecular Probes) and donkey anti-rabbit Alexa Fluor 555 (Molecular Probes) at a 1:2,000 dilution in the same blocking buffer and washed with PBS 3 times. Lastly, the slides were mounted and counter-stained with 40 ,6-diamidino-2-phenylindole dihydrochloride (DAPI, Thermo Fisher) to visualize cell nuclei. The slides were analyzed using a Zeiss Axio fluorescence inverted microscope and Zeiss LSM 710 spectral CLSM microscope (Figure 1). Subretinal Injection

RKO and WT mice were maintained in the breeding colony under cyclic light (200 lux, 12 h light-dark). Bilateral subretinal injections

10

Molecular Therapy Vol. 28 No 2 February 2020

qRT-PCR

Relative qRT-PCR was performed using the StepOnePlus RT-PCR system (Life Technologies Corporation) as described.5,7,69 Trizol Reagent (Life Technologies, Grand Island, NY, USA) was used to extract total retinal RNA according to the manufacturer’s instructions. Briefly, total RNA was extracted from 5 or 6 retinas and 2 mg of isolated RNA was treated with RNase-free-DNase I to remove any remaining gDNA and cDNA that were prepared by reverse transcription using Oligo dT and Superscript III reverse transcriptase (Life Technologies). Non-reverse transcribed RNAs (controls; reactions that contain everything except reverse transcriptase) were included in all experiments to confirm the absence of genomic DNA. qRT-PCR was performed using Sybr green and the CFX96 real-time system (Bio-Rad, Hercules, CA, USA). Each sample was analyzed in triplicate in plates using mouse-specific primer pairs, human-specific primer pairs and mouse/human-specific primers that amplify exons 1 and 2 from the mouse or human transgene, and the primers for the housekeeping gene b-actin. The predicted size of the PCR product and the sequences of the specific primers are listed in Table 1. Relative gene expression was determined according to the DcT method where (DcT = rho cT-b-actin cT). Five to six retinas were assessed for each treatment. To determine the presence of vector genomes, we utilized a pair of primers that amplifies a specific target region between exons 1 and 2 of both mouse and human rhodopsin transgenes, but is mismatched in RKO mice17 (Table 1).

Please cite this article in press as: Zheng et al., Rhodopsin Genomic Loci DNA Nanoparticles Improve Expression and Rescue of Retinal Degeneration in a Model for Retinitis Pigmentosa, Molecular Therapy (2019), https://doi.org/10.1016/j.ymthe.2019.11.031

www.moleculartherapy.org

Western Blot Analysis

Western blot analyses were performed as described previously.6 In brief, 20 mg of lysate protein from primary cultured cells or retinal tissue were separated using a 10% SDS-polyacrylamide gel. The proteins were transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% milk in PBST (1% Tween-20 in PBS) and incubated with the following primary antibodies: mouse rhodopsin monoclonal antibody (mAb) (for human and mouse rhodopsin; 1D4, Santa Cruz Biotechnology), a rabbit anti-human rhodopsin polyclonal antibody (LS-C324699, Lifespan Biosciences), BiP/GRP78 (BD610979), CHOP monoclonal antibody (MA1-250, Thermo Fisher), IRE1a monoclonal antibody (Cell Signaling Tech), and actin monoclonal antibody (Sigma) in 5% milk in PBST overnight at 4 C, then washed 3 times with PBST. The membrane was incubated with appropriate HRP-conjugated secondary antibodies at 1:20,000 for 2 h at RT. The blots were then analyzed using a BioRad Molecular Imager (ChemiDoc XRS system). The densitometric analyses were performed using Image Lab software v4.1 (Bio-Rad). ERG

Full-field ERG was performed to measure scotopic a- and b-wave and photopic b-wave amplitudes as previously described.6,7 Briefly, the eyes of mice that had been dark-adapted overnight were dilated with 1% tropicamide (Bausch & Lomb, Tampa, FL, USA), and kept moist by a drop of GenTeal lubricant eye gel (Alcon, Fort Worth, TX, USA). A single stimulus intensity of 0.01 cd s/m2 (rod-dominated flash), followed by a 2.25 cd s/m2 (rod/cone stimulating flash) and a 30 cd s/m2 flash (cone-dominated flash) was applied to the mice. ERG measurements were made using an Espion E2 system (Diagnosys, Littleton, MA, USA), and body temperature maintained at 37 C. MATLAB software was used to process ERG waveforms. A-waves were measured from baseline to the trough of the downward deflecting wave, whereas the b-wave values were taken from trough of the awave to the peak of upward deflecting wave. At least 6–10 animals were examined per group. Statistical Analyses

GraphPad Prism was used for all statistical analyses. Comparisons between two treatments were performed using a two-tailed Student’s t test. Comparisons between multiple treatments were performed using a one-way or two-way ANOVA as necessary. Data were expressed as means ± SEM (unless otherwise indicated). A value of p <0.05 was considered statistically significant.

SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10. 1016/j.ymthe.2019.11.031.

AUTHOR CONTRIBUTIONS M.Z., R.N.M., and Z.H. designed the project. M.Z., R.N.M., and Z.H. conducted the experiments. M.Z., R.N.M., and Z.H. contributed the protocols and analyzed the data. M.Z., E.R.W., and Z.H. wrote the manuscript. E.R.W. and Z.H. critically reviewed the manuscript. Z.H. supervised the project.

CONFLICTS OF INTEREST The authors declare no competing interests.

ACKNOWLEDGMENTS The authors thank Dr. Alfred Lewin (University of Florida) for critical reading and providing helpful comments and suggestions for the manuscript. This work was supported by the Edward N. & Della L. Thome Memorial Foundation (138289, Z.H.); the BrightFocus Foundation (M2019063, Z.H.); the Carolina Center of Cancer Nanotechnology Excellence (Z.H.); and the U.S. National Eye Institute (R01EY026564, Z.H. and R01EY012224, E.R.W.).

REFERENCES 1. Rogers, S., and Pfuderer, P. (1968). Use of viruses as carriers of added genetic information. Nature 219, 749–751. 2. Han, Z. (2018). Gene therapy using genomic DNA: advances and challenges. In Gene Therapy in Neurological Disorders, M. Li and B.J. Snider, eds. (Elsevier), pp. 63–80. 3. Wade-Martins, R., White, R.E., Kimura, H., Cook, P.R., and James, M.R. (2000). Stable correction of a genetic deficiency in human cells by an episome carrying a 115 kb genomic transgene. Nat. Biotechnol. 18, 1311–1314. 4. Palmiter, R.D., Sandgren, E.P., Avarbock, M.R., Allen, D.D., and Brinster, R.L. (1991). Heterologous introns can enhance expression of transgenes in mice. Proc. Natl. Acad. Sci. USA 88, 478–482. 5. Han, Z., Banworth, M.J., Makkia, R., Conley, S.M., Al-Ubaidi, M.R., Cooper, M.J., and Naash, M.I. (2015). Genomic DNA nanoparticles rescue rhodopsin-associated retinitis pigmentosa phenotype. FASEB J. 29, 2535–2544. 6. Mitra, R.N., Zheng, M., Weiss, E.R., and Han, Z. (2018). Genomic form of rhodopsin DNA nanoparticles rescued autosomal dominant Retinitis pigmentosa in the P23H knock-in mouse model. Biomaterials 157, 26–39. 7. Zheng, M., Mitra, R.N., Filonov, N.A., and Han, Z. (2016). Nanoparticle-mediated rhodopsin cDNA but not intron-containing DNA delivery causes transgene silencing in a rhodopsin knockout model. FASEB J. 30, 1076–1086. 8. Ziady, A.G., Gedeon, C.R., Muhammad, O., Stillwell, V., Oette, S.M., Fink, T.L., Quan, W., Kowalczyk, T.H., Hyatt, S.L., Payne, J., et al. (2003). Minimal toxicity of stabilized compacted DNA nanoparticles in the murine lung. Mol. Ther. 8, 948–956. 9. Yurek, D.M., Fletcher, A.M., Smith, G.M., Seroogy, K.B., Ziady, A.G., Molter, J., Kowalczyk, T.H., Padegimas, L., and Cooper, M.J. (2009). Long-term transgene expression in the central nervous system using DNA nanoparticles. Mol. Ther. 17, 641–650. 10. Han, Z., Conley, S.M., Makkia, R.S., Cooper, M.J., and Naash, M.I. (2012). DNA nanoparticle-mediated ABCA4 delivery rescues Stargardt dystrophy in mice. J. Clin. Invest. 122, 3221–3226. 11. Han, Z., Conley, S.M., Makkia, R., Guo, J., Cooper, M.J., and Naash, M.I. (2012). Comparative analysis of DNA nanoparticles and AAVs for ocular gene delivery. PLoS ONE 7, e52189. 12. Ding, X.Q., Quiambao, A.B., Fitzgerald, J.B., Cooper, M.J., Conley, S.M., and Naash, M.I. (2009). Ocular delivery of compacted DNA-nanoparticles does not elicit toxicity in the mouse retina. PLoS ONE 4, e7410. 13. Kim, A.J., Boylan, N.J., Suk, J.S., Lai, S.K., and Hanes, J. (2012). Non-degradative intracellular trafficking of highly compacted polymeric DNA nanoparticles. J. Control. Release 158, 102–107. 14. Rajala, A., Wang, Y., Zhu, Y., Ranjo-Bishop, M., Ma, J.X., Mao, C., and Rajala, R.V. (2014). Nanoparticle-assisted targeted delivery of eye-specific genes to eyes significantly improves the vision of blind mice in vivo. Nano Lett. 14, 5257–5263. 15. MacLaren, R.E., Pearson, R.A., MacNeil, A., Douglas, R.H., Salt, T.E., Akimoto, M., Swaroop, A., Sowden, J.C., and Ali, R.R. (2006). Retinal repair by transplantation of photoreceptor precursors. Nature 444, 203–207. 16. Zalis, M.C., Johansson, S., and Englund-Johansson, U. (2017). Immunocytochemical Profiling of Cultured Mouse Primary Retinal Cells. J. Histochem. Cytochem. 65, 223–239.

Molecular Therapy Vol. 28 No 2 February 2020

11

Please cite this article in press as: Zheng et al., Rhodopsin Genomic Loci DNA Nanoparticles Improve Expression and Rescue of Retinal Degeneration in a Model for Retinitis Pigmentosa, Molecular Therapy (2019), https://doi.org/10.1016/j.ymthe.2019.11.031

Molecular Therapy

17. Lem, J., Krasnoperova, N.V., Calvert, P.D., Kosaras, B., Cameron, D.A., Nicolò, M., Makino, C.L., and Sidman, R.L. (1999). Morphological, physiological, and biochemical changes in rhodopsin knockout mice. Proc. Natl. Acad. Sci. USA 96, 736–741. 18. Noorwez, S.M., Kuksa, V., Imanishi, Y., Zhu, L., Filipek, S., Palczewski, K., and Kaushal, S. (2003). Pharmacological chaperone-mediated in vivo folding and stabilization of the P23H-opsin mutant associated with autosomal dominant retinitis pigmentosa. J. Biol. Chem. 278, 14442–14450. 19. Lin, J.H., Li, H., Yasumura, D., Cohen, H.R., Zhang, C., Panning, B., Shokat, K.M., Lavail, M.M., and Walter, P. (2007). IRE1 signaling affects cell fate during the unfolded protein response. Science 318, 944–949. 20. Kohli, A., Melendi, P.G., Abranches, R., Capell, T., Stoger, E., and Christou, P. (2006). The Quest to Understand the Basis and Mechanisms that Control Expression of Introduced Transgenes in Crop Plants. Plant Signal. Behav. 1, 185–195.

37. Farjo, R., Skaggs, J., Quiambao, A.B., Cooper, M.J., and Naash, M.I. (2006). Efficient non-viral ocular gene transfer with compacted DNA nanoparticles. PLoS ONE 1, e38. 38. Konstan, M.W., Davis, P.B., Wagener, J.S., Hilliard, K.A., Stern, R.C., Milgram, L.J., Kowalczyk, T.H., Hyatt, S.L., Fink, T.L., Gedeon, C.R., et al. (2004). Compacted DNA nanoparticles administered to the nasal mucosa of cystic fibrosis subjects are safe and demonstrate partial to complete cystic fibrosis transmembrane regulator reconstitution. Hum. Gene Ther. 15, 1255–1269. 39. Ziady, A.G., Gedeon, C.R., Miller, T., Quan, W., Payne, J.M., Hyatt, S.L., Fink, T.L., Muhammad, O., Oette, S., Kowalczyk, T., et al. (2003). Transfection of airway epithelium by stable PEGylated poly-L-lysine DNA nanoparticles in vivo. Mol. Ther. 8, 936–947.

21. Cai, X., Chen, L., and McGinnis, J.F. (2015). Correlation of ER stress and retinal degeneration in tubby mice. Exp. Eye Res. 140, 130–138.

40. Fink, T.L., Klepcyk, P.J., Oette, S.M., Gedeon, C.R., Hyatt, S.L., Kowalczyk, T.H., Moen, R.C., and Cooper, M.J. (2006). Plasmid size up to 20 kbp does not limit effective in vivo lung gene transfer using compacted DNA nanoparticles. Gene Ther. 13, 1048–1051.

22. Awai, M., Koga, T., Inomata, Y., Oyadomari, S., Gotoh, T., Mori, M., and Tanihara, H. (2006). NMDA-induced retinal injury is mediated by an endoplasmic reticulum stress-related protein, CHOP/GADD153. J. Neurochem. 96, 43–52.

41. White, R.E., Wade-Martins, R., and James, M.R. (2002). Infectious delivery of 120kilobase genomic DNA by an epstein-barr virus amplicon vector. Mol. Ther. 5, 427–435.

23. Li, C., Wang, L., Huang, K., and Zheng, L. (2012). Endoplasmic reticulum stress in retinal vascular degeneration: protective role of resveratrol. Invest. Ophthalmol. Vis. Sci. 53, 3241–3249.

42. Fuller, J.A., Shaw, G.C., Bonnet-Wersinger, D., Hansen, B.S., Berlinicke, C.A., Inglese, J., and Zack, D.J. (2014). A high content screening approach to identify molecules neuroprotective for photoreceptor cells. Adv. Exp. Med. Biol. 801, 773–781.

24. Schiedner, G., Morral, N., Parks, R.J., Wu, Y., Koopmans, S.C., Langston, C., Graham, F.L., Beaudet, A.L., and Kochanek, S. (1998). Genomic DNA transfer with a high-capacity adenovirus vector results in improved in vivo gene expression and decreased toxicity. Nat. Genet. 18, 180–183.

43. Gaudin, C., Forster, V., Sahel, J., Dreyfus, H., and Hicks, D. (1996). Survival and regeneration of adult human and other mammalian photoreceptors in culture. Invest. Ophthalmol. Vis. Sci. 37, 2258–2268.

25. Gomez-Sebastian, S., Gimenez-Cassina, A., Diaz-Nido, J., Lim, F., and WadeMartins, R. (2007). Infectious Delivery and Expression of a 135 kb Human FRDA Genomic DNA Locus Complements Friedreich’s Ataxia Deficiency in Human Cells. Mol. Ther. 15, 248–254.

44. Clérin, E., Yang, Y., Forster, V., Fontaine, V., Sahel, J.A., and Léveillard, T. (2014). Vibratome sectioning mouse retina to prepare photoreceptor cultures. J. Vis. Exp. 94, 51954. 45. Bandyopadhyay, M., and Rohrer, B. (2010). Photoreceptor structure and function is maintained in organotypic cultures of mouse retinas. Mol. Vis. 16, 1178–1185.

26. Pérez-Luz, S., Gimenez-Cassina, A., Fernández-Frías, I., Wade-Martins, R., and DíazNido, J. (2015). Delivery of the 135 kb human frataxin genomic DNA locus gives rise to different frataxin isoforms. Genomics 106, 76–82.

46. German, O.L., Buzzi, E., Rotstein, N.P., Rodríguez-Boulan, E., and Politi, L.E. (2008). Retinal pigment epithelial cells promote spatial reorganization and differentiation of retina photoreceptors. J. Neurosci. Res. 86, 3503–3514.

27. Pérez-Luz, S., Abdulrazzak, H., Grillot-Courvalin, C., and Huxley, C. (2007). Factor VIII mRNA expression from a BAC carrying the intact locus made by homologous recombination. Genomics 90, 610–619.

47. Arensdorf, A.M., Diedrichs, D., and Rutkowski, D.T. (2013). Regulation of the transcriptome by ER stress: non-canonical mechanisms and physiological consequences. Front. Genet. 4, 256.

28. Schedl, A., Montoliu, L., Kelsey, G., and Schütz, G. (1993). A yeast artificial chromosome covering the tyrosinase gene confers copy number-dependent expression in transgenic mice. Nature 362, 258–261.

48. Rutkowski, D.T., Wu, J., Back, S.H., Callaghan, M.U., Ferris, S.P., Iqbal, J., Clark, R., Miao, H., Hassler, J.R., Fornek, J., et al. (2008). UPR pathways combine to prevent hepatic steatosis caused by ER stress-mediated suppression of transcriptional master regulators. Dev. Cell 15, 829–840.

29. Kaufman, R.M., Pham, C.T., and Ley, T.J. (1999). Transgenic analysis of a 100-kb human beta-globin cluster-containing DNA fragment propagated as a bacterial artificial chromosome. Blood 94, 3178–3184. 30. Harrington, J.J., Van Bokkelen, G., Mays, R.W., Gustashaw, K., and Willard, H.F. (1997). Formation of de novo centromeres and construction of first-generation human artificial microchromosomes. Nat. Genet. 15, 345–355. 31. Kim, J.H., Kononenko, A., Erliandri, I., Kim, T.A., Nakano, M., Iida, Y., Barrett, J.C., Oshimura, M., Masumoto, H., Earnshaw, W.C., et al. (2011). Human artificial chromosome (HAC) vector with a conditional centromere for correction of genetic deficiencies in human cells. Proc. Natl. Acad. Sci. USA 108, 20048–20053. 32. Basu, J., Compitello, G., Stromberg, G., Willard, H.F., and Van Bokkelen, G. (2005). Efficient assembly of de novo human artificial chromosomes from large genomic loci. BMC Biotechnol. 5, 21. 33. Larin, Z., and Mejía, J.E. (2002). Advances in human artificial chromosome technology. Trends Genet. 18, 313–319. 34. Sgaramella, V., and Eridani, S. (2004). From natural to artificial chromosomes: an overview. Methods Mol. Biol. 240, 1–12. 35. Kouprina, N., Earnshaw, W.C., Masumoto, H., and Larionov, V. (2013). A new generation of human artificial chromosomes for functional genomics and gene therapy. Cell. Mol. Life Sci. 70, 1135–1148. 36. Yurek, D.M., Fletcher, A.M., Smith, G.M., Seroogy, K.B., Ziady, A.G., Molter, J., Kowalczyk, T.H., Padegimas, L., and Cooper, M.J. (2009). Long-term transgene expression in the central nervous system using DNA nanoparticles. Mol. Ther. 17, 641–650.

12

Molecular Therapy Vol. 28 No 2 February 2020

49. Kohno, K., Normington, K., Sambrook, J., Gething, M.J., and Mori, K. (1993). The promoter region of the yeast KAR2 (BiP) gene contains a regulatory domain that responds to the presence of unfolded proteins in the endoplasmic reticulum. Mol. Cell. Biol. 13, 877–890. 50. Mori, K., Sant, A., Kohno, K., Normington, K., Gething, M.J., and Sambrook, J.F. (1992). A 22 bp cis-acting element is necessary and sufficient for the induction of the yeast KAR2 (BiP) gene by unfolded proteins. EMBO J. 11, 2583–2593. 51. Kawai, T., Fan, J., Mazan-Mamczarz, K., and Gorospe, M. (2004). Global mRNA stabilization preferentially linked to translational repression during the endoplasmic reticulum stress response. Mol. Cell. Biol. 24, 6773–6787. 52. Pereira, E.R., Liao, N., Neale, G.A., and Hendershot, L.M. (2010). Transcriptional and post-transcriptional regulation of proangiogenic factors by the unfolded protein response. PLoS ONE 5, e12521. 53. Kroeger, H., LaVail, M.M., and Lin, J.H. (2014). Endoplasmic reticulum stress in vertebrate mutant rhodopsin models of retinal degeneration. Adv. Exp. Med. Biol. 801, 585–592. 54. Gorbatyuk, M.S., Knox, T., LaVail, M.M., Gorbatyuk, O.S., Noorwez, S.M., Hauswirth, W.W., Lin, J.H., Muzyczka, N., and Lewin, A.S. (2010). Restoration of visual function in P23H rhodopsin transgenic rats by gene delivery of BiP/Grp78. Proc. Natl. Acad. Sci. USA 107, 5961–5966. 55. Yang, L.P., Wu, L.M., Guo, X.J., Li, Y., and Tso, M.O.M. (2008). Endoplasmic reticulum stress is activated in light-induced retinal degeneration. J. Neurosci. Res. 86, 910–919.

Please cite this article in press as: Zheng et al., Rhodopsin Genomic Loci DNA Nanoparticles Improve Expression and Rescue of Retinal Degeneration in a Model for Retinitis Pigmentosa, Molecular Therapy (2019), https://doi.org/10.1016/j.ymthe.2019.11.031

www.moleculartherapy.org

56. Tucker, B.A., Mullins, R.F., Streb, L.M., Anfinson, K., Eyestone, M.E., Kaalberg, E., Riker, M.J., Drack, A.V., Braun, T.A., and Stone, E.M. (2013). Patient-specific iPSC-derived photoreceptor precursor cells as a means to investigate retinitis pigmentosa. eLife 2, e00824.

63. Olsson, J.E., Gordon, J.W., Pawlyk, B.S., Roof, D., Hayes, A., Molday, R.S., Mukai, S., Cowley, G.S., Berson, E.L., and Dryja, T.P. (1992). Transgenic mice with a rhodopsin mutation (Pro23His): a mouse model of autosomal dominant retinitis pigmentosa. Neuron 9, 815–830.

57. Kang, M.J., Chung, J., and Ryoo, H.D. (2012). CDK5 and MEKK1 mediate proapoptotic signalling following endoplasmic reticulum stress in an autosomal dominant retinitis pigmentosa model. Nat. Cell Biol. 14, 409–415.

64. al-Ubaidi, M.R., Pittler, S.J., Champagne, M.S., Triantafyllos, J.T., McGinnis, J.F., and Baehr, W. (1990). Mouse opsin. Gene structure and molecular basis of multiple transcripts. J. Biol. Chem. 265, 20563–20569.

58. Wang, F.Y., Jia, J., Song, H.H., Jia, C.M., Chen, C.B., and Ma, J. (2019). Icariin protects vascular endothelial cells from oxidative stress through inhibiting endoplasmic reticulum stress. J. Integr. Med. 17, 205–212.

65. Mitra, R.N., Nichols, C.A., Guo, J., Makkia, R., Cooper, M.J., Naash, M.I., and Han, Z. (2016). Nanoparticle-mediated miR200-b delivery for the treatment of diabetic retinopathy. J. Control. Release 236, 31–37.

59. Saliba, R.S., Munro, P.M., Luthert, P.J., and Cheetham, M.E. (2002). The cellular fate of mutant rhodopsin: quality control, degradation and aggresome formation. J. Cell Sci. 115, 2907–2918.

66. Liu, G., Li, D., Pasumarthy, M.K., Kowalczyk, T.H., Gedeon, C.R., Hyatt, S.L., Payne, J.M., Miller, T.J., Brunovskis, P., Fink, T.L., et al. (2003). Nanoparticles of compacted DNA transfect postmitotic cells. J. Biol. Chem. 278, 32578–32586.

60. Huang, Y., Liu, D.P., Wu, L., Li, T.C., Wu, M., Feng, D.X., and Liang, C.C. (2000). Proper developmental control of human globin genes reproduced by transgenic mice containing a 160-kb BAC carrying the human beta-globin locus. Blood Cells Mol. Dis. 26, 598–610.

67. Han, Z., Guo, J., Conley, S.M., and Naash, M.I. (2013). Retinal angiogenesis in the Ins2(Akita) mouse model of diabetic retinopathy. Invest. Ophthalmol. Vis. Sci. 54, 574–584.

61. Al-Hasani, K., Vadolas, J., Knaupp, A.S., Wardan, H., Voullaire, L., Williamson, R., and Ioannou, P.A. (2005). A 191-kb genomic fragment containing the human alpha-globin locus can rescue alpha-thalassemic mice. Mamm. Genome 16, 847–853.

68. Han, Z., Zhong, L., Maina, N., Hu, Z., Li, X., Chouthai, N.S., Bischof, D., Weigel-Van Aken, K.A., Slayton, W.B., Yoder, M.C., and Srivastava, A. (2008). Stable integration of recombinant adeno-associated virus vector genomes after transduction of murine hematopoietic stem cells. Hum. Gene Ther. 19, 267–278.

62. McNally, N., Kenna, P., Humphries, M.M., Hobson, A.H., Khan, N.W., Bush, R.A., Sieving, P.A., Humphries, P., and Farrar, G.J. (1999). Structural and functional rescue of murine rod photoreceptors by human rhodopsin transgene. Hum. Mol. Genet. 8, 1309–1312.

69. Li, M., Tang, Y., Wu, L., Mo, F., Wang, X., Li, H., Qi, R., Zhang, H., Srivastava, A., and Ling, C. (2017). The hepatocyte-specific HNF4a/miR-122 pathway contributes to iron overload-mediated hepatic inflammation. Blood 130, 1041–1051.

Molecular Therapy Vol. 28 No 2 February 2020

13