**JSRV Intragenic Enhancer Element Increases Expression from a Heterologous Promoter and Promotes High Level AAV-Mediated Transgene Expression in the Lung and Liver of Mice**

#### **Darrick L. Yu, Natalie Chow and Sarah K. Wootton \***

Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada; darrickyu@gmail.com (D.L.Y.); nataliesmchow@gmail.com (N.C.)

**\*** Correspondence: kwootton@uoguelph.ca

Received: 30 September 2020; Accepted: 4 November 2020; Published: 6 November 2020 -

**Abstract:** Jaagsiekte sheep retrovirus (JSRV) induces tumors in the distal airways of sheep and goats. A putative intragenic enhancer, termed JE, localized to the 3′ end of the JSRV *env* gene, has been previously described. Herein we provide further evidence that the JE functions as a transcriptional enhancer, as it was able to enhance gene expression when placed in either forward or reverse orientation when combined with a heterologous chicken beta actin promoter. We then generated novel composite promoters designed to improve transgene expression from adeno-associated virus (AAV) gene therapy vectors. A hybrid promoter consisting of the shortest JE sequence examined (JE71), the U3 region of the JSRV long terminal repeat (LTR), and the chicken beta actin promoter, demonstrated robust expression in vitro and in vivo, when in the context of AAV vectors. AAV-mediated transgene expression in vivo from the hybrid promoter was marginally lower than that observed for AAV vectors encoding the strong CAG promoter, but greatly reduced in the heart, making this promoter/enhancer combination attractive for non-cardiac applications, particularly respiratory tract or liver directed therapies. Replacement of the murine leukemia virus intron present in the original vector construct with a modified SV40 intron reduced the promoter/enhancer/intron cassette size to 719 bp, leaving an additional ~4 kb of coding capacity when packaged within an AAV vector. Taken together, we have developed a novel, compact promoter that is capable of directing high level transgene expression from AAV vectors in both the liver and lung with diminished transgene expression in the heart.

**Keywords:** adeno-associated virus (AAV) vector; jaagsiekte sheep retrovirus (JSRV); LTR; enhancer; transduction

#### **1. Introduction**

Adeno-associated virus (AAV) is a small, non-pathogenic, non-enveloped, single stranded DNA virus belonging to the *Parvoviridae* family [1,2]. In 2017, Luxturna (voretigene neparvovec-rzyl), a recombinant vector based on AAV was approved by the U.S. Food and Drug Administration for the treatment of biallelic RPE65 gene mutation-associated retinal dystrophy, a rare form of inherited vision loss that may result [3]. In 2019, a second AAV gene therapy for the treatment of children less than two years of age with spinal muscular atrophy (SMA), termed Zolgensma, was the second directly administered gene therapy approved in the U.S., demonstrating the growing promise of AAV based gene therapies for the treatment of a wide variety of conditions and inherited disorders.

AAV offers many advantages over other gene delivery vectors such as adenovirus vectors due to its superior transducing efficiency in vivo, its ability to promoter sustained transgene expression, its low immunogenicity and the fact that it can and is being used in a wide range of clinical applications [4]. However, the limited packaging capacity of AAV vectors (~4.7 kb) necessitates the selection of promoter/enhancer elements that are as small as possible, yet retain a high degree of expression, particularly in scenarios where the transgene is of a considerable size, such as in the case of cystic fibrosis transmembrane conductance regulator (CFTR) (4.4 kb). The optimal promoter/enhancer combination for AAV vectored gene therapy applications would be one that has high activity in the target cell population, but minimal to low activity in non-target cells.

Jaagsiekte sheep retrovirus (JSRV) is a simple betaretrovirus that is capable of inducing a form of lung cancer in sheep known as ovine pulmonary adenocarcinoma [5]. Viral gene expression is primarily governed by promoter and enhancer elements located within the long terminal repeat (LTR) sequences that are found on the 5′ and 3′ terminal ends of the integrated provirus [6]. More recently, a putative enhancer sequence, known as JE, has been located outside of the 3′ LTR within the env gene, just prior to the beginning of the 3′ LTR sequence. Adeno-associated virus (AAV) vectors bearing the putative JE enhancer sequence in conjunction with the JSRV long terminal repeat (LTR) demonstrated enhanced, tissue specific expression. Augmentation of the JSRV LTR with the JE resulted in a >4-fold enhancement in lung directed transgene expression and a ~2-fold improvement in liver when vectors were administered to mice [7].

We sought to further delineate the manner in which JE functions and provide further evidence that it is able to function as a transcriptional enhancer. In addition, we hypothesized that promoter/enhancer cassettes based on the JE and the JSRV LTR sequences would be highly effective for in vivo gene delivery purposes, owing to their ability to promote high level protein expression and their relatively compact size.

#### **2. Materials and Methods**

#### *2.1. Cell Culture*

Human embryonic kidney (HEK 293, ATCC CRL-1573) cells, HEK 293T cells, rat fibroblast (208F) cells, and HTX cells, a pseudodiploid subclone of HT-1080 fibrosarcoma cells [8], were maintained in Dulbecco's modified Eagle's medium (DMEM; Thermo Fisher Scientific, Ottawa Canada) supplemented with 10% fetal bovine serum (FBS) (GIBCO, Invitrogen), 100 units/mL penicillin, 100 µg/mL streptomycin and 2 mM L-glutamine in a humidified 5% CO<sup>2</sup> atmosphere at 37 ◦C.

#### *2.2. Molecular Cloning*

PCR was used to amplify JE or LTR sequences from the molecular clone of JSRV, pCMV-JS21 [9]. Vectors encoding human alkaline phosphatase reporter gene (hPLAP) were derived from an AAV vector plasmid, AEEE1AP, as described previously [7]. The vector was modified to replace the Enzootic-Nasal Tumor Virus-1 (ENTV-1) enhancer/promoter component with enhancer elements derived from Jaagsiekte Sheep Retrovirus (JSRV), acting on the chicken beta actin promoter to drive expression of hPLAP. Splicing of the hPLAP encoding transcript was promoted by the presence of a murine leukemia virus env intron [10] found between the enhancer/promoter and the hPLAP gene. Following the hPLAP gene was the SV40 polyA tail for the polyadenylation of transcripts. JE and LTR sequences were cloned into XbaI and BglII sites in the AAV vector plasmid, AEEE1AP [7] containing a murine leukemia virus retrovirus intron and hPLAP reporter gene. The chicken beta actin promoter (CBA) was cloned downstream of the JE or JSRV LTR sequences into BglII and KpnI sites. The sequence for the putative enhancers is as follows: JE71: ACATATGAAATATAGAAATATGTTACAGCACCAACATCTTATGG AGCTTTTAAAAAATAAAGAGAGGGGAG; JE184:ACCCTGATTGGTGTAGGAATACTTGTGTTTAT TATAATTGTCGTAATCCTTATATTTCCTTGCCTTGTTCGTGGCATGGTTCGCGATTTTCTAAAGATG AGAGTTGAAATGCTGCATATGAAATATAGAAATATGTTACAGCACCAACATCTTATGGAGCTTTT AAAAAATAAAGAGAGGGGAG; and JE324: CGTTAGACCTTTTACAACTGCATAATGAGATTCTTG ATATTGAAAATTCGCCGAAGGCTACACTAAATATAGCCGATACTGTTGATAATTTCTTGCAAAA TTTATTCTCTAATTTTCCTAGTCTCCATTCGCTGTGGAAAACCCTGATTGGTGTAGGAATACTT

#### GTGTTTATTATAATTGTCGTAATCCTTATATTTCCTTGCCTTGTTCGTGGCATGGTTCGCGATTTTCT AAAGATGAGAGTTGAAATGCTGCATATGAAATATAGAAATATGTTACAGCACCAACATCTTATGG AGCTTTTAAAAAATAAAGAGAGGGGAG.

#### *2.3. Transfection of Mammalian Cells*

Approximately 5 × 10<sup>6</sup> cells were seeded onto three 10-cm tissue culture dishes for each construct 24 h prior to transfection. Cells were transfected with 10 µg each of pCMV-βgal and the construct of interest using polyethylenimine (Polysciences Inc., Warrington, PA, USA) for 208F rat fibroblast cells and calcium phosphate for human HEK 293, HEK 293T and HTX cells. Polyethylenimine transfection was conducted according to manufacturer's directions. Transfection using the calcium phosphate method was conducted as described previously [11].

#### *2.4. Preparation of Cell Lysates*

After 48 h, cells were washed with 10 mL of ice cold PBS and scraped off the plate using a rubber policeman into 1 mL of PBS. Cells and PBS were spun down at 4000 rpm for 2 min to recover cells. 500 µL of TMNC lysis buffer (50 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 100 mM NaCl, and 4% (wt/vol) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) was added to each cell pellet before pipetting up and down to suspend the cells. Cells were allowed to lyse for 15 min on ice. Cell debris was removed by centrifuging the sample at 14,000 rpm and the supernatant was recovered for use in subsequent assays. A Bradford assay was performed on cell lysates according to the method of Sambrook and Russell to determine total protein concentration [11].

#### *2.5. Beta-Galactosidase Assay*

Beta-galactosidase assays were performed by the method of J. Miller [12]. The following solutions were mixed together prior to performing the assay: 100× Mg2<sup>+</sup> solution containing 0.1 M MgCl<sup>2</sup> and 4.5 M β-mercaptoethanol, 1× ONPG solution containing 4 mg/mL o-nitrophenyl-β-D-Galactoside (ONPG) dissolved in 0.1 M dibasic sodium phosphate buffer pH (7.5). 3 µL of 100× Mg2+, 66 µL of 1× ONPG, 30 µL of cell lysate, and 201 µL of 0.1 M sodium phosphate were mixed together to initiate the reaction. Reactions were incubated at 37 ◦C until a faint yellow color developed. Reactions were stopped by adding 500 µL of 1 M Na2CO3. To determine beta-galactosidase activity, absorbance was read at 420 nm using a BioTeK Powerwave XS2 plate reader.

#### *2.6. In Vivo Administration of AAV Vectors*

Mouse experiments were performed in accordance with the guidelines set forth by the Canadian Council on Animal Care (CCAC). Eight-week old C57BL6/J mice were obtained from Charles River Laboratories (Saint-Constant, QC). AAV vectors were produced by cotransfection of HEK 293 cells with genome and packaging plasmids as described previously [13]. AAV vector titers were determined by quantitative polymerase chain reaction (qPCR) analysis as described elsewhere [14]. AAV vectors were administered via three different routes of administration: intravenous, intraperitoneal, and intranasal to determine relative promoter activity. For intravenous delivery, a phosphate buffered saline (PBS) solution containing 2 × 10<sup>10</sup> vector genomes of AAV vectors was injected in a 100 µL volume into the tail vein. For intraperitoneal, 8 × 10<sup>10</sup> vector genomes were injected into the intraperitoneal space in a 500 µL total volume containing the AAV vector plus PBS. For intranasal delivery, 1 × 10<sup>10</sup> vector genomes were delivered in two aliquots of 40 µL each in order to maximize the chances that tissues deep in the lung would be transduced. A modified method of intranasal delivery was used so as to ensure vector delivery to the distal lung [15]. Mice were euthanized 4 weeks post vector administration, and lungs were perfused through the heart with 20 mL of PBS and then separated into individual lobes. For consistency, the same lung lobe from each mouse was either flash frozen in liquid nitrogen or fixed in 2% paraformaldehyde-PBS for 16 h at 22 ◦C. Half of other major organs, including the liver, spleen, pancreas, nose, heart, and kidney were fixed for 24 h at 22 ◦C, with the other half placed into liquid

nitrogen for a subsequent enzymatic assay of hPLAP activity. Tissues were stained for vector-encoded heat-stable hPLAP as described previously [7]. Gross pictures of stained tissues were taken using a Zeiss dissecting scope (Zeiss Canada, Toronto, ON, Canada).

#### *2.7. Determination of Alkaline Phosphatase Activity*

For in vitro studies, cell lysates were heated at 65 ◦C for 1 h to inactivate endogenous alkaline phosphatases. For 208F cells, 30 µg of total protein as determined by Bradford assay was loaded into each well of a 96 well plate for each 10 cm dish. For HTX, HEK 293, and HEK 293T cells, 100 µg of total protein as determined by Bradford assay were loaded into each well of a 96 well plate. Mouse tissues were harvested 4 weeks after vector administration, snap-frozen in liquid nitrogen and stored at −80 ◦C until assayed. A small piece (approximately 0.5 cm in diameter; ~30 mg) of the tissue to be analyzed was homogenized in TMNC lysis buffer (50 mM Tris HCl pH 7.5, 5 mM MgCl2, 100 mM NaCl, 4% (wt/vol) CHAPS) using a Precellys 24 homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France), with ~200 µL of TMNC buffer in a FastPrep™ Lysing Matrix A tube (MP Bio, Santa Ana, CA, USA). Tissue homogenates were placed in a water bath at 65 ◦C for 1 h to inactivate endogenous heat-labile AP activity and subsequently clarified by centrifugation at 17,900× *g* for 15 min at 4 ◦C to remove cell debris. The protein content of each sample was determined by the method of Bradford, and the AP activity in tissue lysates was determined, in triplicate, by a fluorometric assay using the 4-methylumbelliferyl phosphate (MUP) (Sigma, St. Louis, MO, USA) substrate, as described previously [7]. The mean and standard deviation were calculated for each of the different cell lines and vector constructs, as well as for lung, liver, heart, pancreas and spleen for each of the diffesrent vector treatment groups. To correct for differences in transfection efficiency for each of the plasmid constructs described in Figure 1, the mean of AP activity was divided by the mean of β-gal activity.

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**Figure 1.** Schematic Diagram of Promoter/Enhancer Constructs. (**A**) The structure of an integrated Jaagsiekte sheep retrovirus (JSRV) provirus. A box is drawn around the 3′ end of the *env* gene and the 3 ′ long terminal repeat (LTR) where the putative enhancer lies. (**B**) Promoter/enhancer constructs used to drive human placental alkaline phosphatase reporter gene expression and determine if JE functions as a transcriptional enhancer. JE71, JE184, and JE324 refer to three different lengths of the putative JE enhancer region, 71, 184 and 324 bp in length, respectively. The JE sequences are taken from the 3 ′ end of the *env* gene just prior to the start of the 3′ LTR. In some constructs, the JE was inverted, denoted with an "i". JE and inverted JE were placed in front of the chicken beta actin (CBA) promoter to determine if they could function upon a heterologous promoter. (2) A number of constructs were also evaluated for their utility as gene expression vectors. Constructs bearing the U3 region of the JSRV LTR, the U3 and R region of the LTR, or the full length JSRV LTR (U3, R and U5 regions), in combination with the chicken beta actin promoter were also tested. A subset of these was evaluated in vivo: ACBA-AP, AJE71-CBA-AP, AJE71-U3-CBA-AP, ACAG-AP (a construct bearing the CAG promoter, which combines the CMV immediate early enhancer, chicken beta actin promoter and rabbit β-globin intron). AP denotes the presence of a human placental alkaline phosphatase reporter gene.

#### **3. Results**

#### *3.1. JE on Its Own Functions Poorly as a Promoter*

Constructs bearing short, medium, or long length JE sequences (pAJE71-AP, pAJE184-AP, and pAJE324-AP, respectively) on their own demonstrated little or no expression when transfected into human embryonic kidney (HEK 293), human fibrosarcoma (HTX), or rat fibroblast (208F) cells (Figure 2). Similarly, constructs bearing inverted versions of short, medium, or long length JE sequences (pAiJE71-AP, pAiJE184-AP, and pAiJE324-AP, respectively) also demonstrated low or no expression in these cell lines. The lack of expression for the JE or inverted JE sequences on their own suggest that the JE is unable to function as a promoter in either forward or reverse orientations in these cell lines. Interestingly, there was a low level of activity in HEK 293T cells, but not 293 cells for forward and reverse JE constructs, suggesting that the SV40 large T antigen was able to promote transcription from the AAV2 inverted terminal repeats (ITR) present on the 5′ flanking end of the JE. No SV40 origin of

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replication could be found within the plasmid backbone, suggesting that any increase in expression within 293T cells was independent of plasmid replication by the T antigen. ′

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**Figure 2.** Transfection of four different cell lines (human embryonic kidney (HEK) 293, HEK 293T, rat fibroblast (208F), and human fibrosarcoma (HTX)) with various constructs incorporating the normal orientation JE or the inverted JE. The presence of the inverted JE is denoted by an "i". Three different sizes were compared corresponding to JE sequences 71, 184, or 324 bp in length. These inverted and non-inverted JE sequences were either placed on their own, or in front of the chicken beta actin promoter to determine if the JE could function as a promoter, or in conjunction with a heterologous promoter (chicken beta actin promoter) as an enhancer. The chicken beta actin promoter was also transfected to indicate a baseline level of expression. Normalized Relative Fluorescence Units (RFU) are reported, where AP activity was normalized to β-galactosidase activity. Experiments were conducted in triplicate with three biological replicates.

#### *3.2. JE Enhances Expression from a Heterologous Promoter, in Both Forward and Reverse Orientations, and Extending JE from the 5' End Does Not Appear to Further Increase Expression*

Inclusion of the chicken beta actin promoter (CBA) markedly increased expression from constructs bearing the JE in all cell lines tested (Figure 2). Comparing pAJE71-AP (possessing the short JE, but lacking the chicken beta actin promoter) to pAJE71-CBA-AP (possessing both short JE and the chicken beta actin promoter), a nearly 20-fold increase was observed in 293T cells and an equivalent or greater increase was observed in 293 and HTX cell lines. In a similar manner, comparing pAJE184-AP (possessing medium length JE alone) to pAJE184-CBA-AP (possessing medium length JE plus the chicken beta actin promoter) and pAJE324-AP (possessing the long version of JE alone) to pAJE324-CBA-AP (long JE in addition to the chicken beta actin promoter), there was a ~20-fold increase in expression in 293T cells and a further increase in other cell lines. Extension of the JE from 71 to 184 and 324 base pairs did not appear to confer a corresponding increase in expression, suggesting that the putative enhancer element is located within the original 71 bp region and no additional enhancer elements are located within the 184 or 324 bp regions. A 2-fold or greater increase in transgene expression was also observed when comparing short, medium, or long JE sequences in conjunction with the chicken beta actin promoter relative to the chicken beta actin promoter on its own, demonstrating that it is not merely the chicken beta actin promoter that was able to confer higher expression.

For another series of constructs, the putative JE sequences were placed in an inverted orientation relative to their orientation in the JSRV provirus. Inverted JE sequences corresponding to short, medium and long lengths placed in front of the chicken beta actin promoter (pAiJE72-CBA-AP, pAIJE184-CBA-AP, and pAiJE324-CBA-AP, respectively) all demonstrated a similar improvement as their non-inverted counterparts in 293, HTX, 293T and 208F cells, while constructs containing the inverted JE (pAiJE72-AP, pAiJE184-AP, and pAiJE324-AP) of varying lengths but lacking the chicken beta actin promoter conferred low expression in all cell lines but 293T.

#### *3.3. A Hybrid Promoter Consisting of JE, the U3 and R Regions of the LTR, and the Chicken Beta Actin Promoter Is Highly Active in a Variety of Cell Lines*

The relatively low activity of the wild-type JSRV promoter is dwarfed by the high activity brought about by the combination of the JE, the U3 and R regions of the JSRV LTR placed upstream of the chicken beta actin promoter (pAJE71-U3R-CBA-AP) in 293, 293T, 208F, and HTX cells. In fact, expression was so high in 293T and HTX cells that it matched or exceeded expression by the CAG promoter (pACAGAP), currently one of the best promoters for constitutive expression from a variety of cell lines (Figure 3). A vector consisting of the JE71, U3, but not the R region of the JSRV LTR (pAJE71-U3-CBA-AP) expressed to high levels in the same cell lines, reaching levels comparable to the CAG promoter (Figure 3). Both pAJE71-U3-CBA-AP and pAJE71-U3R-CBA-AP demonstrate expression that was much higher than a vector encoding a promoter consisting of the CBA promoter alone, demonstrating the ability of the JE and JSRV LTR elements to enhance expression from a heterologous promoter.

**Figure 3.** Transfection of four different cell lines (HEK 293, HEK 293T, 208F, and HTX) with various constructs incorporating JE, the chicken beta actin promoter (CBA), and components of the JSRV LTR to determine their suitability for in vivo gene delivery experiments. The JE was investigated to see if it would work with its own homologous promoter, the JSRV LTR, consisting of U3-R-U5 regions, or in conjunction with just the U3/U3 and R regions to enhance expression from the chicken beta actin promoter. These hybrid promoter/enhancer cassettes were compared to the strong CAG promoter and the CBA promoter on its own. Normalized Relative Fluorescence Units (RFU) are reported, where AP activity was normalized to β-galactosidase activity. Experiments were conducted in triplicate with three biological replicates.

Constructs possessing components of the JE or JSRV LTR but lacking the chicken beta actin promoter demonstrated low activity in 293, 293T, 208F and HTX cell lines (Figure 3). pAU3-AP, pAU3-R-U5-AP, pAJE72-U3-R-U5-AP, pAJE184-U3-R-U5-AP, pAJE324-U3-R-U5-AP, pAJE71-U3-AP, and pAJE71-U3-R-AP each exhibited poor expression in 293, 293T, 208F and HTX cells. This may be

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due to the high specificity of JSRV LTR components for tissues of the respiratory tract, the primary target for JSRV infection.

#### *3.4. The JE*/*JSRV LTR*/*CBA Hybrid Promoter Is Active in the Lungs, Nose, Trachea, Liver, Heart, Spleen, and Pancreas of Mice*

Expression of the JSRV promoter is primarily limited to the respiratory tract (lung, nose, trachea) and liver when mice are transduced via an AAV vector [8]. Inclusion of the JE enhancer element appeared to improve expression in these tissues, as well as enhance expression in the spleen and pancreas in mice transduced with AAV vectors via the intranasal, intraperitoneal and intravenous route (Figure 4). A hybrid promoter consisting of the JE71, U3 region of the JSRV LTR, and CBA promoter (AJE71-U3-CBA-AP) was highly active in a variety of tissues when transduced into mice via AAV vectors. High levels of expression could be seen in the tissues of the respiratory tract, including the lung, nose and trachea, as well as the liver, spleen and pancreas both grossly (Figure 4) and histologically (Figure 5). Unlike the JSRV LTR, expression was also observed in the heart, but not to the same extent as the CAG promoter construct, which demonstrated an extremely high level of expression (Figure 4). Note that since the vectors were all packaged into the same AAV capsid, in this case AAV serotype 6 (AAV6), the differences in reporter gene expression observed in vivo are due to the tissue specificity of the promoter and not the capsid. Moreover, the use of a heat stable placental alkaline phosphatase reporter gene (hPLAP) allows for the heat inactivation of all endogenous alkaline phosphatase while retaining hPLAP enzymatic activity. As such, alkaline phosphatase staining, as evidence by the purple color, is only observed if the tissue has been transduced by the AAV vector expressing hPLAP. This can be observed in the mock infected tissues, where no purple staining was detected, either grossly or histologically.

**Figure 4.** Representative gross images of tissues stained to indicate the presence of human placental alkaline phosphatase reporter gene expression from mice transduced with various JE/CBA constructs. AAV vectors were administered to mice by three different routes of administration concurrently: intravenous (2 × 10<sup>10</sup> vg), intraperitoneal (8 × 10<sup>10</sup> vg), and intranasal (1 × 10<sup>10</sup> vg) to determine relative promoter activity. Mice were euthanized 4 weeks post vector administration and tissues stained for human alkaline phosphatase reporter gene (hPLAP) expression.

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**Figure 5.** Representative histological alkaline phosphatase staining of tissue sections from mice transduced simultaneously via the intravenous (2 × 10<sup>10</sup> vg), intraperitoneal (8 × 10<sup>10</sup> vg), and intranasal (1 × 10<sup>10</sup> vg) routes with various AAV vectors bearing JE/CBA promoter/enhancer sequences and imaged 4 weeks post-transduction. Scale bar represents 50 µM.

μ In histological sections, strong AP expression could be observed in lung alveolar cells and liver hepatocytes of mice transduced with a vector that bore the short JE and the CBA (AJE71-CBA-AP), similar to that of ACAGAP (Figure 5). Histological sections of heart transduced with ACAGAP demonstrate very dark staining of individual cells transduced with this vector, compared to the AJE71-U3-CBA-AP, which exhibits a fainter degree of staining (Figure 5). Efficient expression was also observed in pancreatic cells for AJE71-U3-CBA-AP and ACAGAP. Within the spleen, it appeared that no vector was particularly effective.

Quantification of hPLAP enzymatic activity in tissues harvested from the vector transduced mice revealed that in all tissues evaluated, expression from AJE71-U3-CBA-AP was reduced relative to ACAG-AP, but was much higher than ACBA-AP. AJE71-CBA-AP was marginally higher than ACBA-AP but far lower than AJE71-U3-CBA-AP, demonstrating the utility of the JSRV U3 region (Figure 6).

#### *3.5. A Shorter Intron Can Be Used in Conjunction with AJE71-U3-CBA-AP without Reducing Transgene Expression*

The MLV env intron (581 bp) was exchanged for a shorter sequence based on an optimized SV40 intron (93 bp) modified to include consensus splice sites. Transfection of HEK 293 cells with the SV40 intron containing construct compared to the parental plasmid containing the MLV intron demonstrated no visible difference in hPLAP expression (Figure 7).

**Figure 6.** Quantification of alkaline phosphatase activity within homogenized (**A**) lung, (**B**) liver, (**C**) heart, (**D**) spleen and (**E**) pancreas from mice transduced via the intravenous (2 × 10<sup>10</sup> vg), intraperitoneal (8 × 10<sup>10</sup> vg), and intranasal (1 × 10<sup>10</sup> vg) routes with various AAV vectors and harvested for hPLAP enzyme activity analysis 4 weeks post-transduction.

**Figure 7.** Transfection of two different plasmids in HEK 293 cells, utilizing different introns, employing the JE71-U3-CBA promoter. In the image on the left, an intron derived from MLV is used. In the image on the right, a modified SV40 intron was utilized, reducing the size of the promoter–enhancer–intron combination by 488 bp while maintaining a similar amount of expression. Experiments were conducted in triplicate with three biological replicates. Images were taken at 100× magnification.

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#### **4. Discussion**

In its native context, within an integrated JSRV provirus, JE may function to enhance expression from the 5' LTR sequence to drive viral gene expression, or may participate in the process of tumor development by dysregulating the expression of cellular proto-oncogenes.

A lack of expression was observed in constructs bearing only the JE, compared to constructs that possessed the JE and a functional heterologous promoter, the chicken beta actin promoter. Furthermore, inclusion of the JE in any of its varying lengths described here and in either forward or reverse orientations proved to show an increase in protein expression when combined with the chicken beta actin promoter. These characteristics strongly suggest that the JE is able to function as an enhancer sequence [16]. Extension of the JE sequence did not appear to further enhance expression in the cell lines tested, suggesting that no additional enhancer sequences are localized to the area immediately upstream of the 3′ LTR, or at least ones that are active in the cell lines tested. In addition, lengthening of the JE increased the distance between the promoter and inverted terminal repeat of the AAV vector, and no further increase in expression was observed, suggesting that the distance of the promoter from the ITR was not a factor in increasing expression, as previously hypothesized [7]. The particular region where the JE exists overlaps with an RNA export element termed the SPRE or RejRE, which functions in conjunction with a region overlapping the signal peptide of the JSRV envelope to facilitate export of unspliced genomic RNA [17]. However, it is unlikely that the JE functions as an RNA export element in this context as the JSRV envelope is not present in any of the experiments described. Furthermore, there should be no deficit in the ability of transcripts to be exported from the nucleus as they all contain well characterized introns: either the rabbit β-globin intron for the ACAGAP construct or the Moloney murine leukemia virus intron for the other constructs assayed. In addition, the JE sequence should not be transcribed at a high level as it preceded the sequence encoding the promoter in all of the constructs where it was present. Previous attempts at 5′ Rapid Amplification of cDNA Ends (RACE) and RT-PCR were not able to detect the presence of any transcripts except those originating from the R region, when JE71 was placed in front of the JSRV LTR [7].

Low levels of expression observed for the constructs containing JE alone, in either forward or inverted orientation (pAJE71-AP, pAJE184-AP, pAJE324-AP, pAiJE71-AP, pAiJE184AP, and pAiJE324-AP) indicate that JE itself is not able to function effectively as a promoter. This rules out the possibility of JE functioning as a promoter for a transactivating non coding RNA, as is the case in Moloney murine leukemia virus and feline leukemia virus, which both express noncoding RNAs from their 3′ LTRs that are able to transactivate signaling pathways involved in cancer [18–20].

Evidence for a possible expression enhancing region or putative enhancer sequence located in regions flanking the JSRV env gene came to light in a paper by Sinn et al., wherein greatly increased JSRV envelope pseudotyped lentiviral vector titers were observed when env flanking sequences were included in the envelope expression cassette [21]. Previous work with the Prague and Schmidt-Ruppin strains of Rous sarcoma virus has demonstrated that sequences immediately preceding the 3′ LTR are able to enhance expression in reporter gene studies [22,23]. Furthermore, precedence exists for the presence of a region upstream of the 3′ LTR determining the spectrum of disease observed in other retroviruses, such as the exogenous virus-specific region (XSR) sequence of the Prague strain of Rous sarcoma virus, which was demonstrated to be a determinant of oncogenicity [24] and a similar region known as the E region in avian leukosis virus (subgroup J) that has been shown to contribute to oncogenicity in certain chicken breeds [25].

The identity of the transcription factor that binds to the JE has yet to be elucidated; however, one promising candidate is AP-2, for which there are four predicted sites within the JE71 sequence (Figure 8). A wide variety of other transcription factors are also predicted to bind; these include glucocorticoid receptor (GR), for which there are five sites, pituitary-specific positive transcription factor 1 (Pit-1A), for which there are five sites, and c-ETS-2, for which there are four predicted sites. Activation of these pathways through overexpression of transcription factors and/or using pharmacological agents could help to identify which one is responsible for the observed enhancer activity.

′

**Figure 8.** Transcription Factors Predicted to Bind to JE-71. A number of different transcription factors have been predicted to bind to the JE-71 sequence using the EMBOSS 6.5.7 tool tfscan and graphically presented by Geneious. Particularly promising putative transcription factors with multiple predicted binding sites include AP-2 (4 sites), glucocorticoid receptor (GR/GR beta, 5 sites), pituitary-specific positive transcription factor 1 (Pit-1A, 5 sites), and c-ETS-2 (4 sites).

A high level of expression could be observed for AJE71-U3-CBA-AP in vivo, nearly matching ACAGAP for some individual mice (Figures 4–6). However, on an aggregate level, expression was generally lower for all of the organs assayed. Amongst the biggest difference was in the heart, where expression was >2-fold higher in ACAGAP compared to AJE71-U3-CBA-AP. This property might make the JE71-U3-CBA promoter particularly useful when widespread, constitutive expression in a variety of organs is called for, but expression in the heart is to be minimized. The high level of expression observed in the respiratory tract and liver may make this promoter effective for respiratory tract diseases such as lung, paranasal sinus, and nasal cavity cancer, or a monogenic disorder such as cystic fibrosis. At approximately 650 bp in size, JE71-U3-CBA is not particularly large for a promoter, and may be combined with a short intron such as an optimized SV40 intron (93 bp) for a total enhancer–promoter–intron size of approximately 743 bp, leaving ~4 kb for the transgene and polyA signal when used in the context of an AAV vector, which has a coding capacity of ~4.7 kb. This might be attractive for the design of an AAV vector based therapeutic employing the truncated CFTR fragment previously shown to be able to rescue the processing of endogenous F∆508 CFTR in vivo [25]. JE71-U3-CBA and JE71-U3R-CBA enhancer/promoter combinations functioned as effectively as the CAG promoter in the cell lines tested, highlighting the potential of these sequences as alternatives to the commonly used CAG promoter (1621 bp) for in vitro protein expression purposes in mammalian cells.

In summary, we demonstrate the presence of an intragenic transcriptional enhancer element, found within the 3′ end of the env gene. This JSRV-based enhancer element demonstrated strong expression in a variety of tissues, particularly respiratory and hepatic, and promoter/enhancer/intron cassettes derived from these elements can be pared down to a sufficiently small size (<630 bp in length), suitable for genome constraints imposed by AAV vector systems.

**Author Contributions:** Conceptualization, D.L.Y. and S.K.W.; methodology, D.L.Y. and N.C.; formal analysis, D.L.Y. and S.K.W.; writing—original draft preparation, D.L.Y.; writing—review and editing, S.K.W.; supervision, S.K.W.; project administration, D.L.Y.; funding acquisition, S.K.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by NSERC Discovery Grant number RGPIN-2018-04737, Cystic Fibrosis Canada grant ID 3017, and the Mason Research Fund.

**Acknowledgments:** We would like to acknowledge all the veterinary technicians at the Animal Isolation Unit, University of Guelph, for their involvement in the care and maintenance of the animals used in this study.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


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## *Review* **Viral Related Tools against SARS-CoV-2**

**Laura Fernandez-Garcia 1,2,**† **, Olga Pacios 1,2,**† **, Mónica González-Bardanca 1,2 , Lucia Blasco 1,2 , Inés Bleriot 1,2 , Antón Ambroa 1,2, María López 1,2, German Bou 1,2,3 and Maria Tomás 1,2,3,\***


Received: 18 September 2020; Accepted: 15 October 2020; Published: 16 October 2020

**Abstract:** At the end of 2019, a new disease appeared and spread all over the world, the COVID-19, produced by the coronavirus SARS-CoV-2. As a consequence of this worldwide health crisis, the scientific community began to redirect their knowledge and resources to fight against it. Here we summarize the recent research on viruses employed as therapy and diagnostic of COVID-19: (i) viral-vector vaccines both in clinical trials and pre-clinical phases; (ii) the use of bacteriophages to find antibodies specific to this virus and some studies of how to use the bacteriophages themselves as a treatment against viral diseases; and finally, (iii) the use of CRISPR-Cas technology both to obtain a fast precise diagnose of the patient and also the possible use of this technology as a cure.

**Keywords:** SARS-CoV-2; COVID-19; phages; CRISPR; viruses; prevention; diagnosis; treatment

#### **1. Introduction**

At the end of 2019, a new virus appeared in the city of Wuhan (China) and quickly spread throughout the world, causing a global pandemic. The virus is closely related to the SARS-CoV (Severe Acute Respiratory Syndrome Coronavirus), thus named SARS-CoV-2 [1,2]. It is a β-coronavirus, carrying single-stranded, positive-sense RNA genome and four main structural proteins: spike (with two subunits, S1 and S2), envelope, membrane and nucleocapsid (N) [3]. SARS-CoV-2, as SARS-CoV, enters the cell through the receptor-binding domain (RBD) of the S1, which recognizes the angiotensin-converting protein 2 (ACE2), present in the surface of host cells [4]. SARS-CoV-2 provokes COVID-19, a new disease that produces a wide range of symptoms ranging from an asymptomatic carrier state to respiratory distress syndrome and even acute heart injury with the risk of secondary infections [5]. The rapid spread of the virus and the absence of treatment for this new disease have led researchers all over the world to join forces in the search for a solution by using all available resources.

Since the beginning of this pandemic, all the medical resources were focused on two main points: diagnostic and treatment of the disease. The diagnosis of SARS-CoV-2 was firstly based on molecular approaches [6], the real-time RT-PCR assay has become the election method to detect the presence of the virus as it is a specific and sensitive method to disclose viral RNA from respiratory tract samples [7]. In order to establish the presence of the virus, following the WHO's indications, clinical laboratories from all around the world are using various primer pairs: the spike gene, the RNA-dependent RNA

polymerase gene (RdRp), the nucleocapsid gene and the envelope gene [8]. Besides, some serologic analyses have been used to diagnose an active or past infection by quantifying the presence of IgM and IgG in the patient serum [8]. An interesting systematic review and meta-analysis has been carried out concerning the serological assays [9]. Authors concluded that the sensitivity of this technique was higher three weeks after the symptom onset, compared with the first week, and that heterogeneity was found in all analyses. Among the advantages of serological assays, we find that they are cheaper and easier to implement at the point of care, but, above all, they can identify asymptomatic individuals previously infected by SARS-CoV-2. Moreover, serological tests could be deployed as surveillance tools to better understand the epidemiology of SARS-CoV-2. Many serological tests for Covid-19 have become available in a very short period, and this is precisely where their main disadvantage resides: the pace of development of serological tests has been so fast that it has exceeded that of rigorous evaluation. Therefore, uncertainty about the accuracy of serological assays remains important [9].

Concerning the treatment of this disease, and due to its rapid development, finding an effective treatment against it was imperative. Thus, researchers and medical doctors began to test existing medicines and repurposing them as COVID-19 treatments, highlighting: (i) nucleoside analogs, as favipiravir (used for influenza virus, Ebola, chikungunya, yellow fever, enterovirus and norovirus treatment) [10,11], ribavirin (used for treating the respiratory syncytial virus, hepatitis C virus and also against SARS and MERS) [12], remdesivir (used for HIV treatment) [11,13] or galidesivir [14]; (ii) antiparasitics as chloroquine (used against malaria, with positive in vitro results against SARS, MERS, Ebola, HIV, Nipah and Hendra viruses, although no protection was found in vivo against these viruses) [15–18]; (iii) protease inhibitors (lopinavir and ritonavir used as HIV treatments) [19]; (iv) indole-derivate molecules as arbidol (used against hepatitis viruses) [20]; and finally, (v) convalescent plasma therapy from patients who recovered from the infection [21].

Paradoxically, an efficient prevention strategy to combat SARS-CoV-2 could come from different human viruses, e.g., in the form of a vaccine vector. A virus is known as an extremely small infective particle, which can only replicate inside a host. Since their discovery, they have been identified as the cause of a great number of diseases, but more recently, they have also been considered a solution for some of them [22]. Viruses can be genetically modified to express antigens of interest, turning them into efficient vectors that deliver immunogenic particles inside the human body [23]. The usefulness of the viral vectors is based on: (i) their high specificity for their targets, (ii) their ability for gene transduction and (iii) their capacity to generate strong cellular and humoral immune responses without an adjuvant [22]. Besides, all the viruses used as vectors are genetically modified to eliminate their replicative capacity and to decrease or eradicate their pathogenicity. However, a potential problem with viral vectors is the pre-existing immunity, due to previous viral exposure [22].

Indeed, there are viruses able to specifically infect bacteria as well. These are called bacteriophages, and they can also represent an interesting tool useful in the analysis of SARS-CoV-2, in the diagnostic of the disease and in its treatment. Bacteriophages (also known as "phages") are the natural predators of bacteria, highly specific: They recognize the bacterial receptors on the surface of the prokaryotic cells and strongly attach to them [24]. Since the discovery of bacteriophages in 1915 [25], they have been used as an alternative treatment for critical bacterial infections, on some occasions even life-threatening [26,27]. In the last decade, i.e., in the post-antibiotic era, the therapy based on lytic phages (phage therapy) or phage derived proteins (enzybiotics) such as, for instance, phage-encoded endolysins [28], has gained popularity, being one of the few options currently available for infections caused by multi-drug resistant (MDR) bacteria [26,29]. Phages have demonstrated their innocuousness for humans, although some concerns still need to be investigated such as the purity of the preparation [26]. However, this is far from being the only use for phages; they might be a good option to isolate neutralizing antibodies against other infectious diseases, caused by parasites [30] or viruses [31], using the phage display technique.

Highly related to bacteriophages are the Cluster Regulatory Interspaced Palindromic Repeats (CRISPR), discovered in 1993 and firstly named as short regulatory repeats (SRSRs) [32]. It was years later when their function as a bacterial immunity system against bacteriophages was reported [33,34]. CRISPR fragments are phage-derived sequences harbored by bacteria in their chromosomes that act as an acquired immunity system in prokaryotes: when a bacterium that has been infected by a bacteriophage is re-infected by the same type of phage, CRISPR-Cas system recognizes the viral DNA/RNA repeated sequences and digests the spacer segments between the repetitions, using the endonuclease activity of Cas (CRISPR associated) proteins [35]. This system has been extensively studied by many scientific researchers from all over the world and belonging to very different domains [36]. The importance of this technology has been increasing in the last decade, and nowadays, it is even possible to replace one DNA fragment by another; therefore, CRISPR-Cas system is currently considered one of the most important tools to genetic edition, treatment of diseases and genetic modification of mammalian cells, among others [36].

Throughout this work, we have analyzed innovative methods of diagnostic and treatment of this new disease, the COVID-19, based on the use of human viruses, bacterial viruses (bacteriophages), or virus-related tools (CRISPR). Due to the novelty of the topic here discussed and the amount of information available, in this review, some articles that have not been peer-reviewed are cited.

#### **2. Human Viruses as Prevention**

Nowadays, there are several types of viral vectors depending on the type of virus used: retrovirus [37], lentivirus [38], Sendai virus [39], cytomegalovirus [40], poxvirus [41], adenovirus [42], adeno-associated virus (AAV) [43], among others. These vectors have been used against several diseases such as HIV [44–48], hepatitis [49], tuberculosis [50,51], influenza [52,53] and even cancer [54,55]. The most common viruses used for the development of vaccines against human infectious diseases are poxvirus and adenovirus. Poxviruses were the first viruses ever used as vaccine and so the best known with a safety and efficacy widely demonstrated; on the other hand, adenoviruses have been deeply analyzed especially due to its easy production, great transduction efficiency, a broad spectrum of tropism and their transgene expression [22]. The following studies and/or clinical trials measured an elicited humoral response (quantified by ELISA or Western blot) and a neutralizing response (by neutralization assays using either the live virus or a pseudovirus). Neutralizing antibodies can, as their name implies, neutralize the biological effects of the antigen and interfere with their infectivity without a need for immune cells. Currently developed SARS-CoV- and MERS-CoV-specific neutralizing antibodies include monoclonal antibodies (mAbs), their functional antigen-binding fragment (Fab), the single-chain variable region fragment (scFv), or single-domain antibodies. They target S1-RBD, S1-NTD, or the S2 region, blocking the binding of RBDs to their respective receptors and interfering with S2-mediated membrane fusion or entry into the host cell, thus inhibiting viral infections [56].

One of the best-known poxvirus vectors is the Modified Vaccinia Ankara (MVA), unable to replicate in most mammalian cells, thus becoming a safe vector that expresses antigens which elicit an immune response [57]. MVA has been recently modified by Chiuppesi et al. to co-express SARS-CoV-2 spike (S) and nucleocapsid (N) antigens with the aim of testing its immunogenicity and developing a candidate vaccine against COVID-19. In the study, the authors challenged several mice with two MVA vectors, sMVA-S, and sMVA-N vectors, expressing the S and N antigen, respectively. Both vectors were evaluated in a murine model by co-immunization at different doses, and they observed similar SARS-CoV-2 antigen-specific humoral and cellular immune responses in vaccine groups receiving sMVA-S and sMVA-N alone or in combination. Authors claimed that both vectors expressing the S and N antigens can stimulate potent SARS-CoV-2-specific humoral and cellular immune responses in mice, either expressed isolated or in combination. For neutralizing experiments, they used SARS-CoV-2 pseudovirus and detected neutralizing antibodies in all vaccine groups receiving the S antigen. The authors claimed that these neutralizing responses increased after the booster immunization [57].

The adenoviral vector most commonly used for clinical trials and experimental gene therapy applications is HAdV-C5, abbreviated as Ad5 [58]. The research group of Zhu et al. performed a phase-2 trial using a replication-defective Ad5 expressing the spike glycoprotein of SARS-CoV-2, to assess its level of safety, tolerability, and immunogenicity in a group of healthy adults. This trial

did not report serious adverse events within 28 days post-vaccination. They found a peak in specific T-cells at day 14 post-vaccination, whereas the peak in neutralizing antibodies anti-spike occurred at day 28 post-vaccination, detected through both live SARS-CoV-2 virus neutralization and pseudovirus neutralization tests [59].

Nowadays, this vaccine is being tested in humans in a phase-3 clinical trial (Table 1) [60]. Similarly, at the University of Oxford, scientists have designed a chimpanzee adenovirus (ChAdOx1) vectored vaccine encoding a codon-optimized full-length spike protein of SARS-CoV-2 [61]. The authors reported that a single vaccination with ChAdOx1 nCoV-19 was effective in preventing damage to the lungs upon high dose, indicating that vaccination prevents virus replication in the lower respiratory tract, but no reduction in viral shedding from the nose was observed. The biggest limitation of this study was that animals were challenged with a high dose of virus via multiple routes, which does not simulate a realistic human exposure [61]. These researchers performed a phase-1/2 randomized trial in healthy adults and observed that those vaccinated with the ChAdOx1 nCoV-19 (5 × 10<sup>10</sup> viral particles) experimented a few mild/moderate secondary effects during the first days after the vaccination. Nevertheless, authors demonstrated that their vaccine is effective with a single-dose, without several adverse reactions, and detected the presence of high levels of neutralizing antibodies as well as spike-specific antibodies 28 days after vaccination [62]. Currently, this vaccine is in a phase-3 clinical trial in different countries (Table 1) [63].


**Table 1.** Viral-vector vaccine candidates and their current state of development according to the WHO.


**Table 1.** *Cont.*

VLP—Virus-like particle; VSV—vesicular stomatitis virus.

Consistently with their preliminary results, Mercado et al. immunized several rhesus macaques with another adenoviral vector (Ad26) expressing also the spike protein. However, the immunogen that they used was the full-length membrane-bound S protein with a mutation of the furin cleavage site and two proline stabilizing mutations [64]. They reported a robust immune response based on neutralizing antibodies, obtaining complete protection against the SARS-CoV-2 challenge in 5 out of 6 animals [65]. Based on the previous results, they are now performing a phase-1/2 trial in healthy adults in which they are going to administrate two intramuscular doses of the vaccine (Ad26COVS1) [65,66].

In the same context, the Gamaleya Research Institute of Russia had performed two phase-1 clinical trials with adeno-based vaccines. In these clinical trials, they are going to test the safety of two different vaccines, one based in Ad26 and the other in Ad5, both containing the Spike protein of the SARS-CoV-2 and the lyophilizate of the two mentioned above, for the preparation of a solution for intramuscular injection [67,68]. The combination of Ad26 and Ad5 expressing the spike protein is now in phase-3 [67,68].

Moreover, companies from Italy, Germany, and Belgium have joined forces to develop a simian adenoviral vector-based vaccine that expressed the S protein of the SARS-CoV-2, whose phase 1 clinical trial has begun in Italy this summer [69].

Moreover, the Pasteur Institute in collaboration with two companies and the University of Pittsburgh have developed a Measles-vector vaccine expressing a modified surface glycoprotein of the SARS-CoV-2. This vaccine candidate is nowadays in phase 1 clinical trial, to test the safety, tolerability and immunogenicity of a vaccine that is going to be administrated intramuscularly in two doses separated by 28 days in 90 healthy adults [70]. Furthermore, Medicago Inc. has developed another phase-1 trial testing a virus-like particle vaccine that will be injected into healthy adults with or without an adjuvant, trying different doses of the vaccine [71].

Xiamen University is developing an intranasal spray viral-vector vaccine, based on influenza A virus expressing the spike protein (Table 1). This is currently in a phase-1 clinical trial, and the spray is being nasally administered in one dose in 60 healthy adults [72].

Apart from all these clinical trials, according to the World Health Organization (WHO), nowadays there are 49 viral-vector candidates in pre-clinical evaluation: 9 using adenoviruses, 4 using MVA, 7 using influenza A virus, 3 using Measles virus, 5 using VSV (vesicular stomatitis virus), 7 using other viruses, and 12 using virus-like particles [73] (Table 1).

#### **3. Bacteriophages**

As detailed above, bacteriophages are the natural viruses of bacteria that have been used to treat diseases for a long time. Therefore, in 1988, de la Cruz et al. modified the filamentous phage F1 from *Escherichia coli* in order to express repetitive regions from the circumsporozoite protein of *Plasmodium falciparum* [74]. The resulting phages displayed the recombinant protein on their capsid surface and were found to act as carriers capable of producing immunological responses in rabbits. This is one example of how one of the oldest and most abundant entities on Earth has been turned into a powerful therapeutic weapon. Since then, researchers have been investigating the potential of phages in the fight against other infectious diseases. Phage display libraries are a remarkably useful tool that allows the identification of the best ligands for a given target [75], permitting the construction of large libraries consisting of numerous antibody genes [76]. This type of libraries has been used since 1992 to identify specific monoclonal antibodies (mAb) against certain bacteria or viruses [77,78], and a high percentage of human therapeutic antibodies have been developed by this technique [79]. In the past, there have been several examples of phage display libraries expressing viral peptides that have successfully inhibited infections, for instance, the ones caused by adenovirus type 2 [80], hepatitis B virus [81], hantavirus, sin nombre virus [82], and Andes virus [83]. This justifies the use of these libraries as a diagnostic and treatment tools of SARS-CoV-2 (Figure 1).

**Figure 1.** Uses of phage-display libraries in the diagnostic (1, 2 y 3) and treatment (4, 5, and 6) of SARS-CoV-2. scFv: single-chain variable fragment. S2: spike subunit 2. mAbs: monoclonal antibodies. MERS: Middle-East respiratory syndrome. RBD: receptor-binding protein.

#### *3.1. Bacteriophages as Diagnostic Tools: Phage-Display Libraries*

Phage display is a powerful technique for the identification and isolation of peptides or proteins [76]. This technique consists of expressing foreign peptides on the surface of bacteriophages, frequently filamentous bacteriophages isolated from *E. coli*, but not exclusively. Indeed, phagemids are the most commonly used vector in phage display technique: these filamentous-phage-derived vectors contain the replication origin of a plasmid, a selective marker, the intergenic region (usually containing the packing sequence), a gene of a phage coat protein, restriction enzyme recognition sites, a promoter and a DNA segment encoding a signal peptide [84]. Phagemids have small genomes, which makes them suitable to accommodate larger foreign DNA fragments. Moreover, they are more efficient in transformation, which allows for obtaining a phage display library with high diversity. A variety of restriction enzyme recognition sites are available in the genome of phagemids, which is convenient for DNA recombination and gene manipulation. Furthermore, the expression level of fusion proteins can be easily controlled and, finally, phagemids are usually genetically more stable than recombinant phages [84].

Nowadays, the most common phages used are the M13 (*Inoviridae*), T4 (*Myoviridae*), T7 (*Podoviridae*) and Lambda (*Syphoviridae*) [85]. Most of the proteins are displayed as fusion proteins with the Nor C-terminus of different phage surface proteins: coat proteins pIII or pVIII on M13 [86], capsid proteins HOC (highly antigenic outer capsid) or SOC (small outer capsid) on T4 [87], the capsid protein pX on T7 [88] and the head protein pD or the tail protein pV on Lambda phage [89]. To select a specific mAb from the library, phages must be subjected to a multiple-cycle process, and after each one, antibodies showing the highest affinity are chosen for the next round. During each cycle, the library is incubated with the target, previously immobilized on a solid support, washed, eluted, amplified and reselected [90]. In the case of phage display libraries expressing mAbs, these are often quantified by ELISA or similar techniques [91]. In the context of the current COVID-19 pandemic, it is worthy to consider that the use of this method has led to major discoveries concerning highly related coronavirus, like SARS or MERS: (i) identification of two single-chain fragment variable (scFv) antibodies that are highly specific for SARS-CoV [92] and (ii) proposal of a new method, called "Yin-Yang", for selecting

mAbs by using crude antigens of MERS-CoV (Middle East Respiratory Syndrome Coronavirus), which led to the isolation of three mAb against the MERS-CoV nucleocapsid protein [93]. Besides, several researchers have suggested highly specific diagnostic methods that use phage display libraries for the S1 subunit of the SARS-CoV spike protein, with no cross-reaction with other coronaviruses [75].

Phage display libraries have led to several discoveries associated with coronaviruses that have caused serious human diseases in the past, such as SARS-CoV or MERS. One example is the identification of an scFv antibody, called B1, which binds the S2 of the spike protein of the SARS-CoV both in vitro and in vivo, exhibiting a potent neutralizing activity [94]. Moreover, this same technique allowed for the detection of a human Fab (Fragment antigen-binding) molecule against the spike protein of SARS-CoV, named M1A that could be used in passive immunoprophylaxis [95]. However, the Fc (Fragment crystallizable) region of the antibody is needed to enable the development of a proper immune response [96], so structural modifications (as the authors suggested) would be interesting in order to enhance its protective and neutralizing capacities. Following with these libraries, five types of mAb against the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein have been identified [97]. Finally, phage display libraries lead to the identification of two important mAbs, one of which neutralizes the RBD of the SARS-CoV-2 [98] (Figure 1).

#### *3.2. Bacteriophages as Treatments*

In addition to the phage-libraries, other strategies employing phages have been developed as treatments. Regarding the coronavirus type, Ren et al. developed phages that bear specific gastroenteritic coronavirus peptides, which induced humoral and cell-mediated immunity in mice, suggesting that phage-based vaccines may be efficient heterologous antigens for initiating host humoral and cellular immune responses [99]. Furthermore, Lauster et al. modified the icosahedral capsid of Q-beta-phage (Qβ) to display sialic acid ligands that bind to the trimeric haemagglutinin (HA) of the influenza A virus (IAV). These researchers demonstrated that the Qβ-phage capsids can act as highly specific inhibitors of IAV, completely blocking its entry to cells by covering the whole envelope of the virus. However, this method is still undergoing preclinical development [100].

#### **4. CRISPR-Cas**

CRISPR-Cas is a bacterial adaptive immune system that was first demonstrated employing a nuclease enzyme (Cas9) that came in 2007 from Barrangou et al. [34]. However, it was Marraffini et al. who proved, in 2008, that CRISPR did not work by RNA interference but by cutting DNA [101]. In parallel, Deveau and Horvath's groups realized that viral DNA was always digested at the same positions upon infection when the bacterium displayed its CRISPR-Cas immunity system [102,103]. Consequently, they claimed that Cas9 catalyzes the digestion of the DNA at precise positions, encoded by specific sequences of "programmable" RNA (CRISPR-RNA or crRNA), which opened the door to the revolution of CRISPR: a molecular tool that allows accurate site-directed digestion in the DNA. CRISPR can also provide a precise, sensitive diagnostic technique as well as an elegant therapeutic option, which has been applied to identify Zika virus [104], human papillomavirus [105], African Swine Fever virus [106], *Staphylococcus aureus* [107] and *Pseudomona aeruginosa* [108], among others.

#### *4.1. CRISPR-Cas as a Molecular Tool of Diagnostic of COVID-19*

In the last few months, several projects related to CRISPR have appeared or have been modified in response to the current crisis caused by the COVID-19 pandemic [109]. All these techniques use mainly the Cas13 and Cas12 proteins because of their capacity to cut single-strands of either DNA or RNA [110]. Most of CRISPR based techniques have been developed to use LAMP or RT\_LAMP (Reverse transcription loop-mediated isothermal amplification). This technique was developed to simplify the PCR process, with shorter reaction times and no need for specific equipment [111]. Besides, these methods can be developed without high technology or difficulties, allowing the technicians to

perform the diagnostic of the disease directly in the sample collection points. Among this research, we highlight six main diagnostic tests using CRISPR technology (Table 2):



**Table 2.** Comparison of the main characteristics of some novel diagnostic methods for SARS-CoV-2 and the gold standard COVID-19 RT-PCR assay.

<sup>a</sup> This section includes both versions, STOPCovid.v1 and STOPCovid.v2. -: without information.

Recently, Fozouni and collaborators developed an innovative technique based on the direct detection of SARS-CoV-2 from nasal swab RNA extracts using an amplification-free CRISPR-Cas13a-based mobile phone assay. The sensitivity of the technique was around 100 copies/µL and the duration under 30 min, being able to detect a set of positive clinical samples in under 5 min [121].

#### *4.2. CRISPR-Cas as a Treatment*

CRISPR technology has also been proposed as a treatment for IAV and COVID-19 by using the PAC-MAN method (**P**rophylactic **A**ntiviral **C**RISPR in hu**MAN** cells). This system uses the Cas13d protein, which has RNAse activity, to destroy the highly conserved genomic RNA regions of the coronavirus. Cas13d enzyme effectively inhibited and degraded SARS-CoV-2 viral RNA in respiratory epithelial cells. The authors suggest several possible delivery forms for the Cas13d protein and its RNA guides, such as nanoparticles, a DNA-based liposomal strategy and a ribonucleoprotein complex. Furthermore, Cas13d is capable of processing its RNA guides so that multiple RNAs with different targets can be delivered at the same time, thus increasing the chances of complete viral eradication. Although this approach has produced promising results in the laboratory, it is still at the pre-clinical trial stage and must be tested in animal models before being tested in humans [122].

Other authors have suggested AAV as a suitable delivery vehicle for the Cas13d, as each viral particle can pack more than three RNA guides. Moreover, AAVs are excellent, safe delivery systems, and they also have specific lung cell serotypes, enabling administration via the respiratory route. Nevertheless, this delivery method, like the treatment based on CRISPR-Cas13, is still at the pre-clinical trial stage [123].

#### **5. Discussion**

Among this review, we have revised all the viral-based vaccines and viral-related techniques (bacteriophages and CRISPR) that are currently been used in the diagnostic and treatment of SARS-CoV-2. Here, we have summarized all the vaccines that are currently under study (according to WHO), which use viruses as vectors. Moreover, we described the use of phage-display libraries to select monoclonal antibodies specifically against SARS-CoV-2 and how human viruses are used as vectors in vaccines. Finally, in addition to the present techniques, we have reported the new tools that have been developed as new CRISPR diagnosis and treatment methods.

The use of virus-based vaccines has been studied for many years, although until now only one viral-vector vaccine has been approved for use in humans, the rVSV-ZEBOV-GP indicated against Ebola [124]. Nevertheless, some of them are in the final steps of the clinical trials [125]. Despite all the advantages that this kind of vaccines have, they still have some disadvantages such as the pre-existing immunity that can be found against the most common viral-vectors (poxvirus and adenovirus), which might decrease the efficacy of the vaccine [126], or the lack of proper animal models [127]. Nevertheless, in the last months, new murine models have been developed by adding the human ACE-2 receptor to mice by knock-in [128] or transducing the mouse using an adenovirus that expressed the hACE-2 [129]. The viral-vector vaccines are an adequate option in the fight against the COVID-19 disease, being five of the twenty-seven candidate vaccines in a clinical trial to date [73].

However, prophylaxis is not the only way to defeat disease, it is as important to have reliable diagnostic methods and proper treatments. Here we described several new specific diagnostic methods based both in the use of phage-display libraries and CRISPR-Cas. The phage-display libraries are one of the most effective ways to generate a great number of peptides, proteins, or antibodies in a small period [90]. They have been proved useful in the analysis of several autoimmune diseases [130,131] and to produce human antibody therapeutics [132], such as *Helicobacter pylori* [133], *P. aeruginosa* [134], *S. aureus* [135], *Leishmania* [136], Citomegalovirus [137] and Rabies virus [138], among others. However, this technology has a few limitations, like the diversity of the peptides and their quality, which depends on the origin and diversity of the library, as well as on the process employed to evaluate the antibodies [139].

Most of the tools exposed in this review are still under analysis or waiting for their approval, except for the CRISPR-Cas diagnostic systems, some of which are currently accepted with clinical validation. These tools have solved most of the problems that this diagnostic technology had, such as the need for PAM (Protospacer adjacent motif) sequences, quantification of the sample, need to pre-treat the sample or the detection of more than one target per reaction [140]. Interestingly, Fozouni et al. used crRNAs targeting SARS-CoV-2 RNA quantifying viral load using enzyme kinetics, which allows for improvements in the sensitivity and specificity of the diagnosis of COVID19. This innovative assay in combination with mobile phone-based quantification can provide rapid, low-cost, point-of-care screening to aid in the control of SARS-CoV-2 [121]. However, the use of CRISPR technology to treat the disease has to face the main problem of the delivery of the system to the target cells, being proposed several options as phagemids [141] or viruses [142]. Moreover, another problem is the presence of undesirable secondary mutations: although the CRISPR system has a very low frequency of secondary mutations [143], some studies have demonstrated the unnecessary perfect match for the function of the system [144,145].

#### **6. Conclusions**

This work reviews the ultimate tools already developed and in process for the diagnosis and treatment of the new disease COVID-19 using human viruses, bacteriophages, and the bacterial immune system CRISPR-Cas. These methods are the next step in the development of more specific and precise diagnostic tools as well as a new point of view in the treatment of this pandemic, but also useful for many other diseases. Despite the rapid outcome of all the studies presented here, their results leave no doubt about their usefulness against the SARS-CoV-2. They represent an extraordinary opportunity to defeat this disease as well as an incredible example of a common effort of the scientific community all around the world.

**Author Contributions:** Review and writing by L.F.-G. and O.P. (both authors contributed equally to the work); review and correcting by L.B.; review by I.B., A.A., M.L., M.G.-B. and G.B.; supervision, analysis, writing and funding by M.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was funded by grants PI16/01163 and PI19/00878 awarded to M. Tomás within the State Plan for R+D+I 2013–2016 (National Plan for Scientific Research, Technological Development and Innovation 2008-2011) and co-financed by the ISCIII-Deputy General Directorate for Evaluation and Promotion of Research—European

Regional Development Fund "A way of Making Europe" and Instituto de Salud Carlos III FEDER, Spanish Network for the Research in Infectious Diseases (REIPI, RD16/0016/0006) and by the Study Group on Mechanisms of Action and Resistance to Antimicrobials, GEMARA (SEIMC, http://www.seimc.org/).

**Acknowledgments:** Spanish Network for the Research in Infectious Diseases (REIPI, RD16/0016/0006) and by the Study Group on Mechanisms of Action and Resistance to Antimicrobials, GEMARA (SEIMC, http://www.seimc.org/).

**Conflicts of Interest:** All authors declare no conflict of interest.

#### **References**


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