**Analysis of** *CFTR* **Mutation Spectrum in Ethnic Russian Cystic Fibrosis Patients**

#### **Nika V. Petrova \*, Nataliya Y. Kashirskaya, Tatyana A. Vasilyeva, Elena I. Kondratyeva, Elena K. Zhekaite , Anna Y. Voronkova, Victoria D. Sherman , Varvara A. Galkina, Eugeny K. Ginter, Sergey I. Kutsev, Andrey V. Marakhonov and Rena A. Zinchenko**

Research Centre for Medical Genetics, Moskvorechje Street, 1, 115478 Moscow, Russia; kashirskayanj@mail.ru (N.Y.K.); vasilyeva\_debrie@mail.ru (T.A.V.); elenafpk@mail.ru (E.I.K.); elena\_zhekayte@gmail.com (E.K.Z.); voronkova111@yandex.ru (A.Y.V.); tovika@yandex.ru (V.D.S.); vgalka06@rambler.ru (V.A.G.); ekginter@mail.ru (E.K.G.); kutsev@mail.ru (S.I.K.); marakhonov@generesearch.ru (A.V.M.); renazinchenko@mail.ru (R.A.Z.) **\*** Correspondence: npetrova63@mail.ru

Received: 30 March 2020; Accepted: 13 May 2020; Published: 15 May 2020

**Abstract:** The distribution and frequency of the *CFTR* gene mutations vary considerably between countries and ethnic groups. Russians are an East Slavic ethnic groups are native to Eastern Europe. Russians, the most numerous people of the Russian Federation (RF), make about 80% of the population. The aim is to reveal the molecular causes of CF in ethnic Russian patients as comprehensively as possible. The analysis of most common *CFTR* mutations utilized for CF diagnosis in multiethnic RF population accounts for about 83% of all CF-causing mutations in 1384 ethnic Russian patients. Variants c.1521\_1523delCTT (F508del), c.54-5940\_273+10250del21kb (CFTRdele2,3), c.2012delT (2143delT), c.2052\_2053insA (2184insA), and c.3691delT (3821delT) are most typical for CF patients of Russian origin. DNA of 154 CF patients, Russian by origin, in whom at least one mutant allele was not previously identified (164 CF alleles), was analyzed by Sanger sequencing followed by the multiplex ligase-dependent probe amplification (MLPA) method. In addition to the 29 variants identified during the previous test for common mutations, 91 pathogenic *CFTR* variants were also revealed: 29 missense, 19 nonsense, 14 frame shift in/del, 17 splicing, 1 in frame ins, and 11 copy number variations (CNV). Each of the 61 variants was revealed once, and 17 twice. Each of the variants c.1209G>C (E403D), c.2128A>T (K710X), c.3883delA (4015delA), and c.3884\_3885insT (4016insT) were detected for three, c.1766+1G>A (1898+1G>A) and c.2834C>T (S945L) for four, c.1766+1G>C (1898+1G>C) and c.(743+1\_744-1)\_(1584+1\_1585-1)dup (CFTRdup6b-10) for five, c.2353C>T (R785X) and c.4004T>C (L1335P) for six, c.3929G>A (W1310X) for seven, c.580-1G>T (712-1G>T for eight, and c.1240\_1244delCAAAA (1365del5) for 11 unrelated patients. A comprehensive analysis of *CFTR* mutant alleles with sequencing followed by MLPA, allowed not only the identification of 163 of 164 unknown alleles in our patient sample, but also expansion of the mutation spectrum with novel and additional frequent variants for ethnic Russians.

**Keywords:** cystic fibrosis; *CFTR* gene; common and new pathogenic variants; ethnic Russian population

#### **1. Introduction**

Cystic fibrosis (CF, OMIM#219700) is an autosomal recessive condition resulting from the pathogenic variants in the CF transmembrane regulator (*CFTR*) gene. CF is a hereditary disease caused by impaired epithelial chloride channel CFTR function. Variants are classified as disease causing, not disease causing, of variable clinical significance, or of unknown clinical significance. More than 2000 different variants of the *CFTR* gene sequence have been revealed, the pathogenicity of 20% of which is established [1,2]. In many populations the most frequent pathogenic variant of the *CFTR* gene

(*ABCC7*) is F508del, which accounts for approximately two thirds of all *CFTR* alleles, with a decreasing prevalence from Northwest to Southeast Europe. The remaining third of alleles are substantially heterogeneous, with fewer than 20 mutations occurring at a worldwide frequency of more than 0.1%. Some variants can reach a higher frequency in certain populations, due to a founder effect in religious, ethnic or geographical isolates [3]. The spectrum and frequency of *CFTR* gene sequence variants vary significantly in different countries and ethnic groups, which suggests the development of regional molecular diagnostics protocols to optimize medical and genetic care for CF patients [4].

The diagnosis of CF was proven by typical pulmonary or gastrointestinal symptoms or positive neonatal screening, or the diagnosis of CF in a sibling, as well as at least one of the following: two positive sweat chloride tests, or the identification of two *CFTR* pathologic variants in trans according to the guidelines of the European Cystic Fibrosis Society as well as the Russian National Consensus on Cystic Fibrosis [5,6].

Molecular genetic studies on CF have been conducted in the Laboratory of Genetic Epidemiology of the Research Centre for Medical Genetics for a long period of time starting from the year 1989. To date, the laboratory has analyzed the DNA of more than 3400 CF patients, the clinical diagnosis was confirmed in the Scientific-Clinical Department for Cystic Fibrosis of the Research Centre for Medical Genetics. Thereby, 87.4% of the CF patients we examined live in the European part of Russia. More than 85% are Russian or come from marriages between Russians and persons belonging to other ethnic groups. According to the Russian Registry of cystic fibrosis patients of 2017 (RF CF Registry), among at least 212 pathogenic variants of the *CFTR* gene eleven variants are the most frequent ones in the Russian Federation (their relative frequencies exceed 1% in the sample of tested patients) and they are F508del with a share of 52.81%, CFTRdele2,3—6.21%, E92K—3.00%, 2143delT—2.15%, 3849+10kbC>T—2.02%, W1282X—1.90%, 2184insA—1.85%, 1677delTA—1.81%, N1303K—1.54%, G542X—1.35%, and L138ins with 1.24% [7]. All other *CFTR* variants identified in Russian patients share 12.35%. The frequencies and spectrum of variants of the *CFTR* gene vary in different regions. This is caused by specific ethnic background of the population, as well as by different population processes occurring on different territories inhabited by the same ethnos. Thus, in the North Caucasus Federal District (NCFD), three variants are the most frequent ones: F508del (25.0%), 1677delTA (21.5%), and W1282X (17.2%) [7]. A study of *CFTR* gene variants' spectra in different NCFD ethnic groups revealed a high proportion of variant W1282X (88%) for Karachays [8], and variants 1677delTA (81.5%) and E92K (12.5%) for Chechens [9]. The most frequent variants in the Volga Federal District (VFD) are F508del (50.5%), E92K (8.7%) and CFTRdele2,3 (5.0%) [7]. A high share of E92K variant in VFD is due to the prevalence of this variant for Chuvash (55%) [10]. The second most frequent variant for Chuvash CF patients is F508del (30%) [9], although this value is lower than in the total sample of CF patients (according to the Registry of CF patients in the Russian Federation 2017, [7]).

Russian East Slavic ethnos is the most numerous people in the Russian Federation (RF) (more than 111,000,000 people), which makes 77.7% of the population of the country according to census of 2010 [11]. In the European part of RF, Russians make 85%–90% of the population.

The aim is to describe the Russian-specific spectrum of pathogenic variants of the *CFTR* gene, testing of which could increase the informativeness of DNA diagnostics in regions with a predominantly Russian population, as well to establish a basis for forming a patient base for possible targeted therapy.

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

Initially, *CFTR* genotyping of 1384 CF patients (ethnic Russians) from all-Russian sample (3457 CF patients) tested in the Laboratory of Genetic Epidemiology, Research Centre for Medical Genetics were analyzed. The diagnosis of CF was made in the Scientific-Clinical Department for Cystic Fibrosis, Research Centre for Medical Genetics or in regional CF centers according to the accepted standards [10]. Diagnosis was confirmed by analysis of clinical presentation and Gibson–Cooke sweat test, with chloride ion concentrations of 60 mmol/L or higher defining positive result. The assignment of patients' Russian ancestry was based on self- or parents' reports. The study included 154 CF Russian patients, 90% of whom came from the European part of the Russian Federation and 10% from Siberian or Far Eastern regions, for all of them at least one mutant allele was not identified.

Patients or their parents signed an informed consent to the study. The research protocol was approved by the Ethical Committee of Research Centre for Medical Genetics (Research Centre for Medical Genetics, 115522, Moscow, Moskvorechie St., 1, Russian Federation, Protocol No.17/2006 of 02.02.2006).

Molecular diagnostics consists of three consecutive stages.

First stage included analysis of 33 frequent *CFTR* variants (c.54-5940\_273+10250del21kb (p.Ser18Argfs\*16, CFTRdele2,3), c.254G>A (p.Gly85Glu, G85E), c.262\_263delTT (p.Leu88IlefsX22, 394delTT), c.274G>A (p.Glu92Lys, E92K), c.350G>A (p.Arg117His, R117H), c.413\_415dupTAC (p.Leu138dup; L138ins), c.472dupA (p.Ser158LysfsX5, 604insA), c.489+1G>T (621+1G>T), c.1000C>T (p.Arg334Trp, R334W), c.1040G>C (p.Arg347Pro, R347P), c.1397C>G (p.Ser466X, Ser466X), c.1519\_1521delATC (p.Ile507del, I507del), c.1521\_1523delCTT (p.Phe508del, F508del), c.1545\_1546delTA (p.Tyr515X, 1677delTA), c.1585-1G>A (1717-1G>A), c.1624G>T (p.Gly542X, G542X), c.1652G>A (p.Gly551Asp, G551D), c.1657C>T (p.Arg553X, R553X), c.2012delT (p.Leu671X, 2143delT), c.2051\_2052delAAinsG (p.Lys684SerfsX38, 2183AA>G), c.2052\_2053insA (p.Gln685ThrfsX4, 2184insA), c.2657+5G>A (2789+5A>G), c.3140-16T>A (3272-16T>A), c.3476C>T (p.Ser1159Phe, S1159F), c.3475T>C (p.Ser1159Pro; S1159P), c.3535\_3536insTCAA (p.Thr1179IlefsX17, 3667ins4), c.3587C>G (p.Ser1196X, S1196X), c.3691delT (p.Ser1231ProfsX4, 3821delT), c.3718-2477C>T (3849+10kbC-T), c.3816\_3817delGT (p.Ser1273LeufsX28, 3944delGT), c.3844T>C (p.Trp1282Arg, W1282R), c.3846G>A (p.Trp1282X, W1282X), c.3909C>G (p.Asn1303Lys, N1303K), representing a routine Russian Federation panel that identifies up to 85% of mutant CF alleles as described previously [12].

Second stage included analysis of *CFTR* gene coding sequence, exon-intron junctions and 5- -UTR sequence by Sanger sequencing as described previously [12]. Variant pathogenicity status (only pathogenic or likely pathogenic variants were reported) was established using consensus recommendations of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology for interpretation of sequence variants and Russian recommendations. The frequencies of identified alleles in general populations were established based on the GnomAD browser (https://gnomad.broadinstitute.org/). The predicted functional effect of missense variants was determined through SIFT, FATHMM and Radial SVM prediction algorithms as well as GERP++ and PhyloP conservation scores. Intronic and splicing variants were analyzed using Human Splicing Finder tool v. 2.4.1. Novel variants were submitted to the CFTR2 website dataset (https://cftr2.org/), CFTR1 (http://www.genet.sickkids.on.ca/cftr). Pathogenic variants of the *CFTR* gene are denoted according to the legacy nomenclature, besides novel variants named according to the HGVS nomenclature for NM\_000492.4 (*CFTR*) transcript variant.

Third stage intended to search for large rearrangements in chromosome region 7q31.2 (deletions/duplications–CNV) involved the *CFTR* gene locus by the multiplex ligation-dependent probe amplification (MLPA) method in case when no pathogenic allele was detected or an allele with uncertain significance was identified at the previous stages. MLPA analysis was performed with SALSA MLPA probemix P091-D2 CFTR (MRC-Holland, Amsterdam, the Netherlands) according to the manufacturer's recommendation. The MLPA results were analyzed using Coffalyser.Net (MRC-Holland) [12].

Variants phase was checked by segregation analysis in proband and healthy parents.

The gIVS6a\_415\_IVS10\_987Dup26817bp (CFTRdup6b-10) duplication and its boundaries were previously described by F.M. Hantash and co-authors [13]: The fragments duplicated started 415 bp downstream of exon 6a, in IVS6a, and spanned exons 6b, 7, 8, 9, and 10, breaking at 2987 bp downstream of exon 10 in IVS10. The duplicated region is 26,817 bp. Two pairs of primers have been developed to clarify the boundaries of CFTRdup6b-10 duplications identified in Russian CF patients. One flanks the junction area of rupture points of intron 11 (10 as in the legacy nomenclature) and intron 6a (6): IVS10F 5- -TCAGGAAATGGCAATGGGGT-3 and IVS6aR 5- -GGCTCTGGTGTGATGATCCATA-3- . A 359 bp fragment from these primers is amplified only from the allele carrying the duplication. The second pair (INT10F 5- -GGGGTTGGGAAGTGATTCCA-3 and INT10R 5- -GCCATCAGCTAGGCTTCTGTA-3- ) flanks the rupture area of the intron 10, amplification occurs only from the normal sequence of the intron 10 of the *CFTR* gene leading to a product of 234 bp.

To compare variant frequencies, the Fisher test was used. The significance level was considered to be *p* ≤ 0.05.

#### **3. Results**

When developing a routinely used mutation panel, the laboratory's own data [14], the results of the first collaborative study [15], and studies of other Russian laboratories (in St. Petersburg [16], Bashkortostan [17], and Tomsk [18]) were considered. The panel includes 33 pathogenic variants of the *CFTR* gene identified in patients from different regions of the Russian Federation, as well as the variants specific for certain ethnic groups [7–10], and allows identification of up to 85% of mutant alleles in all-Russian population [12].

At the first stage, the results of testing 33 pathogenic variants of the *CFTR* gene in DNA of 1384 ethnic Russians with CF (previously performed in the laboratory of genetic epidemiology) were analyzed. Thereby, 29 out of 33 tested variants were revealed (Table 1). In addition to F508del and CFTRdele2,3, eight more variants can be referred to as frequent ones for ethnic Russians (frequency of variants 2143delT, 3849+10kbC-T and 2184insA exceed 2%, variants N1303K, G542X, E92K, W1282X, and L138ins exceed 1%). The mutation detection rate of the used panel of tested variants is 83% in the sample of ethnic Russians (Table 1). In 932 patients, two mutant variants were identified, in 426 patients only one pathogenic variant was detected, both alleles were not detected in 26 patients.


**Table 1.** Frequencies of 33 variants of *CFTR* gene in a sample of 1384 ethnic Russians and in a nationwide sample of CF patients (RF CF Registry) [7].

On the second stage, 154 ethnic Russians affected by CF, for whom one or both mutant alleles were not identified when analyzing 33 mutations, were selected from the sample of 1384 CF patients for further analysis. Their genotypes were presented in Supplementary Table S2. There was a total of 164 unidentified mutant alleles of the *CFTR* gene.

As a result, in addition to 29 identified frequent mutations, 91 pathogenic (or likely pathogenic) genetic variants in the *CFTR* gene were detected (Table 2). Of these, 29 are missense mutations, 19 nonsense mutations, 14 frame-shift mutations (11 deletions and three insertions)), 17 splice-site, one in-frame insertion, 11 large rearrangements (eight deletions and three duplications).



*Genes* **2020**, *11*, 554


**Table 2.** *Cont*.

#### *Genes* **2020** , *11*, 554


**Table 2.** *Cont*.

(i)–in-frame insertion, (CNV)–copy number variation (large

rearrangement).

#### *Genes* **2020**, *11*, 554

#### **4. Discussion**

At present, routine DNA testing of patients includes analysis for 33 pathogenic variants in the *CFTR* gene (Materials and methods section). The spectrum of variants included in the first stage of molecular genetic research was developed gradually. Therefore, 1384 ethnic Russian CF patients are included in the present study, for whom all 33 variants have been tested.

The choice of the spectrum of mutations for routine analysis is conditioned by the results obtained in the course of our own studies [14], studies conducted in different laboratories of the Russian Federation on independent samples of CF patients [6,12,16,17], and data on the prevalence of *CFTR* gene mutations in a global sample of CF patients published by the World Health Organization [18] and presented in the *CFTR* mutation database CFTR1 [19].

Mutations c.1521\_1523delCTT (F508del), c.1624G>T (G542X), c.1652G>A (G551D), c.1657C>T (R553X), c.3846G>A (W1282X), c.3909C>G (N1303K), c.489+1G>T (621+1G>T), c.350G>A (R117H), and c.1585-1G>A (1717-1G>A), are among ones the most common in the world [18,20]. Therefore, first of all these mutations were included in the analysis of Russian patients. Variants c.1519\_1521delATC (I507del), c.254G>A (G85E), c.3718-2477C>T (3849+10kbC-T), c.1000C>T (R334W), and c.1040G>C (R347P), although not among the most common in the world, are quite common for many populations with specific ethnic background. In 1993–1995, in order to detect pathogenic variants specific to the Russian population, a joint study of the coding sequence of the *CFTR* gene was carried out with the Institute of Biogenetics (Brest, France) by denaturing gradient gel electrophoresis with subsequent sequencing in a sample of 50 patients. It was shown that, in addition to the previously detected *CFTR* gene mutations, the mutations c.2012delT (2143delT), c.2052\_2053insA (2184insA), c.262\_263delTT (394delTT), and c.3691delT (3821delT) can also be considered frequent for ethnic Russian CF patients. [15]. In the collaborative study of Dörk T. with co-authors [21], in which our laboratory also participated, the predominant distribution of mutation c.54-5940\_273+10250del21kb (CFTRdele2,3) was shown for the populations of Eastern Europe and the relative frequency of this mutation was determined for the studied Russian patients (7.2%); second in frequency after the mutation c.1521\_1523delCTT (F508del). T.E. Ivashchenko [16] for the first time describes the variants c.1545\_1546delTA (1677delTA), c.3587C>G (S1196X) and c.3844T>C (W1282R), relatively frequent for CF patients from Russia. The variant c.1545\_1546delTA (1677delTA) was shown to be common for Georgian patients, whereas variants c.3587C>G (S1196X) and c.3844T>C (W1282R) were identified for Russian CF patients. In a study conducted in our laboratory, variants c.3535\_3536insTCAA (3667ins4), c.3816\_3817delGT (3944delGT), c.472dupA (604insA), and c.413\_415dupTAC (L138ins) were identified and included in the frequent mutations' panel [14].

#### *4.1. Similarity and Di*ff*erence of Frequency Profiles of Common CF Variants in Two Samples of Russian Patients and the Data of CFTR2*

A comparison of frequency profiles of 33 variants tested at the first stage shows similarity of frequency distributions for ethnic Russian patients and for patients of All-Russian sample (Table 1): the most frequent is c.1521\_1523delCTT (F508del) (54.99% and 52.81%, respectively), the second in frequency is c.54-5940\_273+10250del21kb (CFTRdele2,3) (7.59% and 6.21%), and frequencies of eight more variants exceed 1%. This similarity is not surprising, as ethnic Russians make up the majority (over 85%) of CF patients in the Russian Federation. However, there also are differences. Frequencies of the variants c.1521\_1523delCTT (F508del) (*p* = 0.059), c.54-5940\_273+10250del21kb (CFTRdele2,3) (*p* = 0.018), c.2012delT (2143delT) (*p* = 0.109), c.3718-2477C>T (3849+10kbC>T), c.2052\_2053insA (2184insA), c.3909C>G (N1303K), c.1624G>T (G542X), c.3844T>C (W1282R), c.1397C>G (pSer466X), c.3691delT (3821delT), c.3816\_3817delGT (3944delGT) are higher for ethnic Russian patients' sample than for the all-Russian one (Table 1, Figure 1). Perhaps, this is due to the fact that these variants are typical for ethnic Russians and may reflect this ancestry. While frequencies of other variants prevail in the all-Russian sample, which reflects the fact that these variants prevail among patients belonging to other ethnic groups. Thus, the frequency of variant c.1545\_1546delTA (1677delTA) for ethnic Russians is much lower than for the all-Russian sample (0.18% and 1.81%, respectively, *p* < 0.0001). The variant c.1545\_1546delTA (1677delTA) is predominantly distributed in the North Caucasus populations (Chechens, Ingush, Kumyks) [9,12]. The frequency of variant c.262\_263delTT (394delTT) for ethnic Russians is lower than for the all-Russian sample (0.54% vs. 0.94%, *p* = 0.074, although difference is not significant). In the Russian Federation, it is more often found among the population associated with the past settlement of the Finno-Ugric peoples in northwestern European regions and in the Volga-Ural region [12,17]. The frequency of variant c.274G>A (E92K) for ethnic Russian patients is almost three times less than for the all-Russian sample (1.05% vs. 3.00%, *p* < 0.0001). The frequency of variant c.274G>A (p.Glu92Lys, E92K) is maximum for Chuvash (up to 55%) [10], high for Tatars (6.67%), Bashkirs (6.25%) [7], Chechens (12.5%) [9]. The frequency of c.3846G>A (p.Trp1282X, W1282X) is significant higher in the all-Russian sample (RF CF Registry) than in ethnic Russian patients (1.16% vs. 1.90%, *p* = 0.012).

**Figure 1.** Frequency distribution of *CFTR* gene variants in three samples of CF patients: ethnic Russians, CF RF Registry, CFTR2 database [22].

When comparing CF-causing variant frequencies in ethnic Russian CF patients to the CFTR2 database [22], significant frequency difference was found (Fig. 1, Supplementary Table S1). So, the frequencies of c.54-5940\_273+10250del21kb (p.Ser18Argfs\*16, CFTRdele2,3), c.2012delT (p.Leu671X, 2143delT), c.3718-2477C>T (3849+10kbC-T), c.2052\_2053insA (p.Gln685ThrfsX4, 2184insA), c.274G>A (p.Glu92Lys, E92K), c.413\_415dupTAC (p.Leu138dup; L138ins) and some other variants appear higher in ethnic Russian patients while the frequencies of c.1521\_1523delCTT (p.Phe508del, F508del), c.1624G>T (p.Gly542X, G542X), c.2657+5G>A (2789+5A>G), c.489+1G>T (621+1G>T), c.1657C>T (p.Arg553X, R553X), c.254G>A (p.Gly85Glu, G85E), c.1040G>C (p.Arg347Pro, R347P), c.350G>A (p.Arg117His, R117H) were lower. Variants c.3844T>C (p.Trp1282Arg, W1282R), c.3140-16T>A (3272-16T>A), and c.3816\_3817delGT (p.Ser1273LeufsX28, 3944delGT) were not listed in CFTR2. Variants c.1652G>A (p.Gly551Asp, G551D), c.1585-1G>A (1717-1G>A), and c.3476C>T (p.Ser1159Phe, S1159F) were not found in tested cohort of Russian patients (Supplementary Table S1). However, the differences in frequencies in these latter series involve rare variants and their significance remains unknown.

#### *4.2. Sanger Sequencing Detection of the CFTR Gene Variants*

As a result of analysis of the coding sequence and regions of exon-intron junctions 80 variants in addition to preliminary tested common *CFTR* gene variants were identified. 61 variants identified in this work were identified on one chromosome and 17 on two chromosomes (Table 2). Each of the variants c.1209G>C (E403D), c.2128A>T (K710X), c.3883delA (4015delA) and c.3884\_3885insT (4016insT) were detected for three, c.1766+1G>A (1898+1G>A) and c.2834C>T (S945L) for four, c.1766+1G>C (1898+1G>C) and c.(743+1\_744-1)\_(1584+1\_1585-1)dup (CFTRdup6b-10) for five, c.2353C>T (R785X) and c.4004T>C (L1335P) for six, c.3929G>A (W1310X) for seven, c.580-1G>T (712-1G>T) for eight, and c.1240\_1244delCAAAA (1365del5) for 11 unrelated patients (Table 2).

Some of genetic variants identified in sequencing were first discovered in this study. Description of 15 is presented in a previously published paper [23]. Nine of these variants are nonsense mutations (c.252T>A (p.Tyr84X), c.831G>A (p.Trp277X), c.1083G>A (p.Trp361X), c.3112C>T (p.Gln1038X)) or frame-shift mutations (c.264\_268delATATT (p.Leu88PhefsX21), c.1219delG (p.Glu407AsnfsX35), c.1608delA (p.Asp537ThrfsX3), c.1795dupA (p.Thr599AsnfsX2), c.3189delG (p.Trp1063X), resulting in the formation of premature stop codon (Table 1). Variant c.490-1G>C breaks the acceptor site of 5 exon splicing. These variants belong to the category of PVS1 null variants (pathogenic variant sequence) according to the criteria of classification of pathogenicity of genetic variants [24]. Variant c.1792\_1793insAAA (p.Lys598dup) leads to the insertion of lysine into position 598, and clinical significance of the variant is assessed as pathogenic. The clinical significance of the missense mutations (c.358G>C (p.Ala120Pro), c.1382G>A (p.Gly461Glu), c.1513A>C (p.Asn505His), c.1525G>C (p.Gly509Arg)) is assessed as probably pathogenic.

Eight more variants are presented for the first time. Two variants-nonsense mutations (c.1204G>T (p.Glu402X), c.2617G>T (p.Glu873X)) and one deletion with frame shift (c.2312delA (p.Asn771ThrfsX2)) are concluded to be PVS1 null variants according to the ACMG classification. Variant c.2989-2A>C is a violation of the 19 exon splicing site. Three missense mutations (c.613C>A (p.Pro205Thr), c.1352G>T (p.Gly451Val), c.1589T>C (p.Ile530Thr), c.3107C>A (p.Thr1036Asn)), the clinical significance of which is assessed as probably pathogenic according to the recommendations [24]. The characteristics of the phenotypes of patients who carry rare missense variants are presented in Supplementary Table S3.

#### *4.3. CNV in Russian CF Patients Detected by MLPA*

Large rearrangements of the *CFTR* gene were found for 18 unrelated patients, which is 10.8% (18/166) of the tested mutant alleles and should account for about 1% in the total sample of all mutant alleles in Russians. The MLPA method revealed 11 large rearrangements of the *CFTR* gene: three duplications and eight deletions (Table 1). Four of the large rearrangements were detected in several families. Thus, the duplication of a fragment covering 7–11 (6b–10) exons was detected for five unrelated patients. The testing system we developed allowed us to confirm that the duplication detected had the same frames as previously described in the literature [13]. In the RF CF Registry 2017, this variant was noted for six more unrelated patients. Thus, CFTRdup6b-10 was detected in eleven unrelated patients. Six of them live in the Volga-Ural region, three in the Central region. It should be noted that two patients from the Volga-Ural region belong to the other ethnic groups: one-Bashkir and one-Udmurt.

Each of the deletions, c.(53+1\_54-1)\_(164+1\_165-1)del (CFTRdele2), c.[(1679-1\_1680+1)\_(2490+1 \_2491-1)del[;](2908+1\_2989-1)del] (CFTRdele12,13;del16) and c.(273-1\_274+1)\_(869+1\_870-1)del(1209-1 \_1210+1)\_(1392+1\_1393+1)del (CFTRdel4-7;del9-10) was detected twice. Complex deletion, CFTRdele12,13;del16, was detected for two patients from unrelated families living in the Moscow region; deletion CFTRdel4-7;del9-10 for two families from the Kaliningrad region and the Republic of Buryatia; deletion CFTRdele2 in families from the Transbaikal region and Irkutsk region.

#### *4.4. Detection Rate of Three-Stage Analysis of CFTR Gene in Russian CF Patients*

As a result of analysis of the coding sequence and regions of exon-intron junctions and subsequent search for large rearrangements, 163 out of 164 alleles were identified that were not detected after preliminary testing of frequent variants of the *CFTR* gene.

In one patient only variant E217G with the F508del in trans was detected after sequencing and MLPA. In NCBI-ClinVar database variant E217G is considered to be variant of conflicting interpretation of pathogenicity (benign; likely benign; uncertain significance) [25]. In the study by Lee J.H. et al. [26] it was shown that non-synonymous E217G mutation in the M470 background caused a 60%–80% reduction in *CFTR*-dependent Cl<sup>−</sup> currents and HCO3 − transport activities. So we might suggest that the clinical presentation in that patient is due to complex allele E217G-M470 (Supplementary Table S3).

The second mutant allele of the *CFTR* gene could not be identified in one sample. Failure to identify the second pathologic mutation in the *CFTR* gene after sequencing the coding sequence and searching for large rearrangements may be due to the location of the pathogenic variant either in inner regions of the introns, or in regulatory regions of the *CFTR* gene, or in regulatory regions outside the *CFTR* gene. Indeed, such variants have been recently identified, for example, c.1680-883A>G, c.2989-313A>T, c.3469-1304C>G, or c.3874-4522A>G, that lead to the creation of a new donor splice site and the activation of a cryptic acceptor splice site, resulting in the inclusion of an additional pseudo-exon (PE) and the loss of wild type (WT) CFTR transcripts [27].

#### **5. Conclusions**

In a representative sample of CF patients (ethnic Russians), the spectrum of 33 routinely analyzed (in Russia) variants of the *CFTR* gene was studied. It was shown that, out of 29 identified variants, frequencies of only 10 exceed 1%, and the mutation detection rate of testing did not exceed 85%. Consistent use of sequencing and MLPA methods has allowed us to identify a significant variety of *CFTR* gene mutations spectrum (91 additional genetic variants), to expand the spectrum of frequent variants (c.1766+1G>C (1898+1G>C), c.2353C>T (R785X), c.(743+1\_744-1)\_(1584+1\_1585-1)dup (CFTRdup6b-10), c.4004T>C (L1335P), c.3929G>A (W1310X), c.580-1G>T (712-1G>T), c.1240\_1244delCAAAA (1365del5), detected for five and more unrelated patients, to increase the detection rate of identified mutant alleles for Russian CF patients up to 99.4%, consistently using the strategy of Sanger sequencing and MLPA analysis. This information can be useful for the further optimization of medical genetic counseling in CF high-risk families, for improving the neonatal screening program for CF, and for making decision about the possible CFTR modulators therapy in the future. The identification of previously unknown CF-pathogenic or likely-pathogenic variants is a useful piece of information for diagnostic testing not only in Russia, but worldwide, and can be considered as a contribution to the general knowledge about the *CFTR* variant heterogeneity.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2073-4425/11/5/554/s1, Table S1: Frequencies of 33 variants of *CFTR* gene in the samples of 1384 ethnic Russians and in the CF patients from CFTR2 database [22], Table S2: Genotypes and *CFTR* gene variants in 154 Russian patients tested for 33 common *CFTR* variants, Table S3: Clinical and demographic characteristics of CF patients with rare missense variants.

**Author Contributions:** Data curation, N.V.P. and N.Y.K.; Formal analysis, N.V.P. and T.A.V.; Funding acquisition, S.I.K. and R.A.Z.; Investigation, N.V.P.; Project administration, E.K.G., S.I.K. and R.A.Z.; Resources, N.Y.K., E.I.K., E.K.Z., A.Y.V., V.D.S. and V.A.G.; Supervision, E.K.G. and R.A.Z.; Validation, T.A.V. and A.V.M.; Writing–original draft, N.V.P.; Writing–review & editing, A.V.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** The research was partially supported by grant RFBR 20-015-0061A and within the state task of the Ministry of education and science of Russia.

**Acknowledgments:** The authors are grateful to Stanislav Krasovsky and Elena Amelina for their input to the collection of the clinical data of adult CF patients. Also the authors would like to thank the National CF Patient Registry for providing access to patients data and thank the individual regional CF centers representatives for allowing the use of data, https://mukoviscidoz.org/mukovistsidoz-vrossii.html.

**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**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Establishment of a Recombinant AAV2**/**HBoV1 Vector Production System in Insect Cells**

**Xuefeng Deng 1, Wei Zou 1, Ziying Yan <sup>2</sup> and Jianming Qiu 1,\***


Received: 27 March 2020; Accepted: 15 April 2020; Published: 17 April 2020

**Abstract:** We have previously developed an rAAV2/HBoV1 vector in which a recombinant adeno-associated virus 2 (rAAV2) genome is pseudopackaged into a human bocavirus 1 (HBoV1) capsid. Recently, the production of rAAV2/HBoV1 in human embryonic kidney (HEK) 293 cells has been greatly improved in the absence of any HBoV1 nonstructural proteins (NS). This NS-free production system yields over 16-fold more vectors than the original production system that necessitates NS expression. The production of rAAV with infection of baculovirus expression vector (BEV) in the suspension culture of Sf9 insect cells is highly efficient and scalable. Since the replication of the rAAV2 genome in the BEV system is well established, we aimed to develop a BEV system to produce the rAAV2/HBoV1 vector in Sf9 cells. We optimized the usage of translation initiation signals of the HBoV1 capsid proteins (Cap), and constructed a BEV Bac-AAV2Rep-HBoV1Cap, which expresses the AAV2 Rep78 and Rep52 as well as the HBoV1 VP1, VP2, and VP3 at the appropriate ratios. We found that it is sufficient as a trans helper to the production of rAAV2/HBoV1 in Sf9 cells that were co-infected with the transfer Bac-AAV2ITR-GFP-luc that carried a 5.4-kb oversized rAAV2 genome with dual reporters. Further study found that incorporation of an HBoV1 small NS, NP1, in the system maximized the viral DNA replication and thus the rAAV2/HBoV1 vector production at a level similar to that of the rAAV2 vector in Sf9 cells. However, the transduction potency of the rAAV2/HBoV1 vector produced from BEV-infected Sf9 cells was 5–7-fold lower in polarized human airway epithelia than that packaged in HEK293 cells. Transmission electron microscopy analysis found that the vector produced in Sf9 cells had a high percentage of empty capsids, suggesting the pseudopackage of the rAAV2 genome in HBoV1 capsid is not as efficient as in the capsids of AAV2. Nevertheless, our study demonstrated that the rAAV2/HBoV1 can be produced in insect cells with BEVs at a comparable yield to rAAV, and that the highly efficient expression of the HBoV1 capsid proteins warrants further optimization.

**Keywords:** rAAV2/HBoV1; baculovirus; insect cells

#### **1. Introduction**

Adeno-associated virus (AAV) and human bocavirus (HBoV) are members in different genera of the parvovirus family [1]. AAV is a nonpathogenic parvovirus and its productive replication needs the function of a helper virus [2,3]. In contrast, HBoV1 is a human pathogen that causes lower respiratory tract infections in young children worldwide [4–12]. In vitro, HBoV1 infects only polarized human airway epithelium cultured at an air–liquid interface (HAE-ALI), and replicates autonomously [13–16]. While both are nonenveloped, small, single-stranded (ss) DNA viruses, AAV packages both the plus- and minus-strand genome with equal efficiency, whereas HBoV1 prefers

packaging the minus-strand [14]. HBoV1 has a genome of 5543 nucleotides (nts) in length, which is 18.5% (864 nts) larger than the AAV2 genome of 4679 nts [14].

The genome organizations of these two viruses are quite different. HBoV1 uses one promoter to express all viral nonstructural (NS) and structural or capsid (Cap) proteins, but AAV uses three different promoters [4,17]. The coding sequence of AAV consists of two large open reading frames (ORFs) encoding the nonstructural or replication (Rep) proteins and the Cap proteins at the left and right half of the AAV genome, respectively [18]. The large Rep78/68 and the small Rep52/40 proteins are expressed from the viral mRNAs that are transcribed by an upstream promoter at unit 5 of the genome (P5) and an internal promoter (P19), respectively. Three AAV Cap proteins, VP1, VP2 and VP3, are expressed from the mRNA transcribed by the P40 promoter [2,19]. In addition, a small NS protein, assembly-associated protein (AAP), is alternatively translated from the P40-transcribed Cap-coding mRNA [20], which plays a role in capsid assembly [21–24]. Recently, another small NS protein, membrane-associated accessory protein (MAAP), has been identified, which is also expressed from the P40-transcribed mRNA through alternative translation [25]. It exists in all AAV serotypes and was believed to play a role in the life cycle of AAV. HBoV1 expresses five NS proteins, NS1, NS2, NS3, NS4, and nuclear protein (NP1), and three Cap proteins, VP1, VP2, and VP3, as well as a bocaviral noncoding small RNA (BocaSR) [26–29]. The middle ORF, which is located at the center of the genome between the left ORF encoding NS1-4 and the right ORF encoding VP1-3, encodes NP1 [4]. NP1 plays an important role in viral DNA replication [30], as well as in regulation of HBoV1 cap expression [28,31].

The sequences at both termini of AAV and HBoV1 contain important motifs that are necessary for viral genome replication and virion assembly. In AAV, they are inverted terminal repeats (ITRs) [2,19], but in HBoV1 they are asymmetric with a non-perfectly palindromic hairpin at the left end terminus and a perfectly palindromic hairpin at the right terminus [14]. The transfection of the plasmid clone of a complete AAV genome in human embryonic kidney (HEK)293 cells leads to the production of AAV virions, but this only occurs in the presence of infection of a helper virus, such as adenovirus or co-transfection of a plasmid helper harboring all the adenoviral helper genes (*E2*, *E4Orf6*, and *VA RNA*) [32]. While HEK293 cells are not permissive to HBoV1 infection, the transfection of the cloned HBoV1 genome can produce HBoV1 infectious virions. The progeny virions infect HAE-ALI with a robustly high efficiency, even at a multiplicity of infection (MOI) of at 0.001 viral genome per cell [14,15].

Trans-complementation supports the replication and package of the gutless rAAV2 genome containing only cis elements of their termini and a gene of interest, which have been effectively developed as rAAV vectors for gene therapy of genetic diseases [2,19,33]. The safety profiles of the rAAV genome have been proven from tremendous preclinical studies and clinical trials of human gene therapy [34–41]. Up to date, two rAAV-based drugs, Luxturna and Zolgensma, have been approved by the US FDA. Similarly, a recombinant HBoV1 vector (rHBoV1) was produced in HEK293 cells via trans-complementation [42]. rHBoV1 efficiently transduced HAE-ALI from the apical membrane; however, the safety concern of being derived from a human pathogen limits its application. To overcome this disadvantage for a safe vector to transduce human airway epithelium from the airway lumen with the emphasis on gene therapy for cystic fibrosis (CF) lung disease, we successfully developed a cross-genera chimeric parvovirus vector, rAAV2/HBoV1 [42], in which the safety-proven rAAV2 genome was packaged into the airway tropic HBoV1 capsid. Importantly, the rAAV2/HBoV1 expands the package capacity of the rAAV2 genome by 20%, up to 5.8-kb [42]. Apical application of an rAAV2/HBoV1 carrying a full-length CF transmembrane conductance regulator (CFTR) cDNA of 5.4-kb to CF HAE-ALI cultures, which were made of primary airway epithelial cells of CF patients, efficiently corrected CFTR-dependent chloride transport [42]. In addition, the rAAV2/HBoV1 vector efficiently transduced ferret airways in vivo [43]. Therefore, it holds much promise for gene delivery to human airways, as well as for preclinical trials of CF gene therapy using CF ferret models [44]. Recently we have increased the production efficiency of the rAAV2/HBoV1 vector in HEK293 cells through

optimization of cap expression, which approaches a similar level of rAAV2 production in HEK293 cells [45]. However, a robust vector production system is in demand for future CF gene therapy in preclinical and human trials using the rAAV2/HBoV1 vector.

Traditional rAAV vector productions utilize HEK293 cells. During the rAAV2 or the rAAV2/HBoV1 production in HEK293 cells, the rescue and replication of the rAAV2 genome require the expression of AAV *rep* in addition to the adenoviral helper genes [19,46]. rAAV2 can also be produced in insect cells by the infection of baculovirus expression vectors (BEVs). The AAV-BEV production system represents a robust and scalable bioprocess [47–52], which only requires one of the large Rep78/68 and one of the small Rep52/40 [53]. AAP is required for efficient production of certain serotypes of rAAV vectors in Sf9 cells [54,55]. Co-infection of BEVs—one carrying an rAAV2 genome and one expressing AAV2 Rep78 and Rep52 along with AAV2 VP1, VP2, and VP3—has been used to produce the rAAV vector in a large quantity at a yield of up to ~105 copies per Sf9 cell, compared to the yield of ~103 copies per HEK293 cell [47,53,54,56].

In this report, we explored the possibility of rAAV2/HBoV1 vector production in the BEV system. Our study demonstrated that the rAAV2/HBoV1 vector can be efficiently produced in a suspension Sf9 culture. In the presence of the expression of HBoV1 NP1, a vector yield similar to that of rAAV2 was achieved in Sf9 cells. To our knowledge, this is the first report that the parvoviral cross-genera pseudopackage is also effective in insect cells.

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

#### *2.1. Cell and Cell Culture*

Human embryonic kidney (HEK) 293 cells: HEK293 cells (CRL-1573), obtained from American Type Culture Collection (ATCC; Manassas, VA, USA), were cultured in Dulbecco's modified Eagle's medium (GE Healthcare Life Sciences, Piscataway, NJ, USA) with 10% fetal bovine serum (#F0926, MilliporeSigma, St. Louis, MO, USA)

Insect cells: Sf9 cells (CRL-1711, ATCC) were cultured in suspension in SFX-Insect medium (GE Healthcare, Marlborough, MA, USA) with 2% fetal bovine serum (#F0926, Millipore Sigma; St. Louis, MO, USA) at 27 ◦C.

HAE-ALI cultures: primary human airway cells were isolated from human lung tissues, and this procedure was carried out at the Tissue and Cell Culture Core of the Center for Gene Therapy, University of Iowa. The primary cells were cultured in the airway basal cell expansion medium (#CC-3118, Lonza, Basel, Switzerland), supplemented with 10 μM of ROCK inhibitor Y-276322, 1 μM of A8301, 1 μM of DMH-1, and 1 μM of CHIR99021 (Tocris Biosciences, Minneapolis, MN, USA) until confluency [57]. Then, the cells were collected and seeded onto collagen-coated inserts (Transwells; #3470, Corning, Tewksbury, MA, USA) with a density of 50,000 cells/well. After cell attachment for two days, the apical and basolateral medium were removed and replaced with a complete Pneumacult-ALI medium (StemCell, Vancouver, Canada) at the basolateral chamber to initiate an airway–liquid interface. The medium was changed every two days, and the ALI-cultured HAE took about four weeks to be fully differentiated. We monitored the cultures with a transepithelial electrical resistance using an epithelial Ohmvoltmeter (Millicell-ERS; EMD-Millipore, Burlington, MA, USA), and only HAE-ALI cultures with a resistance value of over 1000 <sup>Ω</sup>·cm2 were used for subsequent transduction.

#### *2.2. Construction of Baculoviral Expression Shuttle Plasmids and Other HEK293 Cell-Expressing Plasmids*

pFastBacDual(m): the plasmid pFastBacDual (Invitrogen, Carlsbad, CA, USA) was modified by inserting a 0.83-kb fragment of λ DNA which contains a SbfI site at each end through SnaBI at nt 3983 and MfeI at nt 4815 to obtain the plasmid pFastBacDual (m).

pBac-AAV2ITR-GFP-Luc (5.4-kb): this BAC-AAV transfer plasmid was constructed by replacing the 0.83-kb λ DNA in pFastBacDual(m) with a 5444-nt ITR-flanked (rAAV) proviral DNA into the at two SbfI sites (Figure 1A). The intermediate rAAV proviral plasmid pAAV-CMV(P10)-GFP-SV40- Luc-bGHpA was derived from pAAV-MCS vector (Cell Biolabs, Inc., San Diego, CA, USA). Foreign

DNA flanking with a pair of SbfI sites was cloned into EcoRI and BamHI digested pAAV-MCS, including the P10 promoter, an open reading frame (ORF) of an enhanced green fluorescent protein (GFP; excised from pEGFP1, Clontech, Palo Alto, CA, USA), SV40 polyadenylation signal (polyA), SV40 early promoter, a FLAG-tagged ORF of firefly luciferase (Luc) and a stuffer from λ DNA (to make the rAAV2 genome of 5444-nt).

**Figure 1.** Construction of baculoviral transfer plasmids for vector production in Sf9 cells and the plasmids for rAAV2/HBoV1 vector production in HEK293 cells. (**A**) BEVs for rAAV2/HBoV1 production. Schematically diagrammed are structures inside the BEVs that were involved in rAAV2/HBoV1 production. Bac-AAV2ITR-GFP-Luc carries an rAAV2 genome of 5.4-kb; Bac-AAV2Rep-HBoV1Cap expresses AAV2 Rep proteins and HBoV1 capsid proteins as shown; and Bac-HBoV1NP1 expresses HBoV1 NP1. P10 and Ph are baculoviral promoters, and CMV and SV40 are cytomegaloviral immediate early and SV40 virus early promoters, respectively. PolyA: polyadenylation signal; Luc: firefly luciferase. (**B**) Plasmids used for vector production in HEK293 cells. pAAV2ITR-GFP-Luc carries the same rAAV2 genome as shown in panel A. pCMVNS\*Cap-P5Cap is a two-in-one plasmid. It was derived from the plasmid pHBoV1CMVNS\*Cap [28], in which the NS1/2 ORF was early terminated. An AAV2 P5 and P19 driven *rep* gene was cloned after the CMV promoter-driven HBoV1 *cap* gene expression cassette. (**C**,**D**) Codon optimization. Both wild type (wt) and optimized (opt.) sequences between ATGs of the AAV2 Rep78 and Rep52 ORFs (**C**) and of the HBoV1 VP1 and VP3 ORFs (**D**) are diagrammed. Nucleotides in red indicate mutations. (**E**) BEVs for rAAV2 production in Sf9 cells. Bac-AAV2ITR-GFP carries a GFP expression cassette under both the CMV and P10 promoters. Bac-AAV2Rep-Cap carries expression cassettes of AAV2 *cap* and AAV2 *rep* under the P10 and Ph promoters, respectively.

pBac-AAV2Rep-HBoV1Cap: to obtain a modified AAV2 *rep* gene expression cassette of a bifunctional Rep78- and Rep52-encoding mRNA, we synthesized a 637-bp DNA fragment containing a partially codon-optimized (opt)Rep78 ORF [56] (Figure 1C), and amplified the full-length optRep78/52 ORF using overlapped PCR, which was cloned into pFastBacDual through BglII (BamHI)-XbaI sites and resulted in pFastBacDual-AAV2Rep. We also synthesized a fragment of 390-bp containing an optimized HBoV1 sequence between VP1 AUG and VP3 AUG [28,58], as shown in Figure 1D, and amplified the full length optVP1/2/3 ORF using overlapped PCR, which was then cloned into the pFastBacDual-AAV2Rep through XhoI-NheI sites to obtain the BEV transfer plasmid pBac-AAV2Rep-HBoV1Cap (Figure 1A).

pBac-HBoV1NP1: HBoV1 NP1 ORF was cloned into pFastBacDual between the XhoI-KpnI sites to get the transfer construct pBac-HBoV1-NP1 (Figure 1A).

pAAV2ITR-GFP-Luc: to parallel compare the capability of the Sf9 cell and HEK293 cell systems, we made a plasmid, pAAV2ITR-GFP-Luc (Figure 1B), based on the backbone of pAAV-MCS promoterless (Cell Biolabs). This was achieved by cloning the fragments from the plasmid of pFastBacDual(m)-AAV2-ITR-GFP-Luc through two NotI sites.

pCMVNS\*Cap-P5Rep: this was the HBoV1 *cap* and AAV2 *rep* expression two-in-one helper plasmid (Figure 1B), which was constructed by cloning the P5- and P19-driven AAV2 *rep* expression cassette from the pAV-Rep2 plasmid [42] into the pCMVNS\*Cap [28], which expresses HBoV1 capsid proteins under the cytomegalovirus immediate early promoter (CMV) through a SacII site.

All the plasmids were sequenced to confirm the expressing genes and the critical elements for virus production at MCLAB (South San Francisco, CA, USA).

#### *2.3. Recombinant Baculovirus Expression vector (BEV) Production*

BEVs were generated by transfection of the BEV shuttle plasmid into DH10BacTM *E. coli* competent cells, following the instructions of the Bac-to-Bac Baculovirus Expression System (Invitrogen, Carlsbad, CA). Bac-AAV2ITR-GFP (Bac-GFP) [47] and Bac-AAV2Rep-Cap (Bac-RepCap2) [56] (Figure 1E) were obtained from The University of Iowa Viral Vector Core Facility. BEVs were titrated in plaque forming units (pfu) by a plaque assay as described in the manual of the Bac-to-Bac Baculovirus Expression System (Invitrogen) or quantified using quantitative real-time PCR (qPCR) with an amplicon targeting to the gentamicin-resistance gene (Probe: 5- 6FAM-ACA TTC ATC GCG CTT GCT GCC TTC-3- ZEN /Iowa Black FQ; forward primer: 5- -CGG GAA CTT GCT CCG TAG TAA-3- , and reverse primer: 5- -CGC CAA CAA CCG CTT CTT-3- ).

#### *2.4. rAAV vector Production*

For production of AAV vectors in insect cells, 200 mL of Sf9 cells in suspension culture at a density of 2 <sup>×</sup> 10<sup>6</sup> cells/mL were co-infected with BEVs at an MOI of ~10 (pfu/cell). At 72 hrs post-infection, cells were collected by centrifugation and lysed in phosphate-buffered saline, pH7.4 (PBS). After four times of freezing-thawing, the cells were sonicated at the setting of 70% power for 3 min (1 min sonicate with 1 min of interval), followed by DNase I treatment at 37 ◦C for 45 min and 10% deoxycholate with 0.25% Trypsin-EDTA incubation at 37 ◦C for another 30 min. Then, CsCl was added into cell crude lysate at a final concentration of 0.472 g/mL and incubated at 37 ◦C for 30 min. The mixture was centrifuged at 3,500 rpm for 30 min, the clarified virus/CsCl solution was transferred into another tube and adjusted to a final density of about 1.40 g/mL. The final clear mixture was loaded into tubes and centrifuged at 41,000 rpm for 36 hrs at 20 ◦C using an TH641 rotor in a Sorvall™ WX (Thermo Scientific). After two rounds of CsCl banding, an aliquot (500 μL) of gradient fractions was collected using a Gradient Station (BioComp Instruments, Fredericton, N.B., Canada), determined for values of refractive index using an Abbe Refractometer, and quantified by qPCR for vector genomes. The peak fractions were dialyzed against PBS buffer.

For the HEK293 cell system, the rAAV2/HBoV1 vector was generated by co-transfection of pAAV2ITR-GFP-Luc, pCMVNS\*Cap-P5Rep, and pHelper that expresses adenoviral E2, E4Orf6 protein and VA RNA [3] into twenty 150-cm<sup>2</sup> plates of HEK293 cells (80% confluency) at a ratio 2:3:1, as previously described [42]. At 72 hrs post-transfection, cell pellets were collected and treated, and recombinant vector was purified as described above for infected Sf9 cells.

#### *2.5. Western Blot and Southern Blot Analyses*

Western blotting was performed as previously described [59]. For Southern blotting, low molecular weight (Hirt) DNA was extracted from BEV infected Sf9 cells, and the analysis was performed as previously described [60], using an AAV2 *cap* gene probe.

#### *2.6. Quantitative Real Time PCR (qPCR) Analysis of rAAV2*/*HBoV1*

The titers of rAAV2 and rAAV2/HBoV1 in DNase I digestion-resistant particles (DRP) were determined by a qPCR method that has been used previously [42]. Briefly, 50 μL aliquots of the samples were incubated with 20 units of Benzonase (MilliporeSigma) for 2 hrs at 37 ◦C, followed by 20 μL of proteinase K (15 mg/mL) at 56 ◦C for 10 min. Viral DNA was extracted using a QIAamp blood mini kit (Qiagen, Hilden, Germany), and then eluted in 50 μL of deionized water. A plasmid containing a GFP ORF was used to establish a standard curve for absolute quantification. The amplicon primers and dual-labeled probe were designed using Primer Express (Applied Biosystems, Foster City, CA, USA) and synthesized at IDT Inc. (Coralville, Iowa, USA). The sequences of the primers and probe specific to the GFP coding sequence are as follows: forward primer, 5- -CTG CTG CCC GAC AAC CA-3- ; reverse primer, 5- -TGT GAT CGC GCT TCT CGT T-3- ; and dual-labeled probe, 5- 6FAM-TAC CTG AGC ACC CAG TCC GCC CT-3- Iowa Black FQ. Probe qPCR MasterMix (Applied Biological Materials Inc., Vancouver, Canada) was used for qPCR following a standard protocol on a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), and 2 μL of extracted DNA was used in a reaction volume of 20 μL.

#### *2.7. Transmission Electron Microscopy*

For each recombinant virus, aliquots of 50 μL in the peak fractions were performed for electron microscopy analysis in the Electron Microscopy Research Laboratory (EMRL) of the University of Kansas Medical Center. Briefly, two to five μL of each sample were spotted onto formvar-coated, carbon-stabilized, 200-mesh copper grids for 1 min and then washed with deionized water. Staining was achieved by adding five drops of 2% uranyl acetate. Excess staining was removed immediately by adsorption to filter paper, and the samples were then air dried. The grids were examined on a Transmission Electron Microscope (JEM-1400; JEOL, Peabody, MA, USA) at a magnification setting of 30,000 × and an accelerating voltage of 100 KV.

#### *2.8. rAAV2*/*HBoV1 Transduction of HAE-ALI Cultures*

We followed a previously described protocol to infect HAE-ALI cultures with rAAV2/HBoV1 [42]. Briefly, proteasome inhibitor doxorubicin and N-acetyl-l-leucyl-l-leucyl-l-norleucine (LLnL) at the final concentrations of 2.5 μM and 20 nM, respectively, were added into the culture medium of the basolateral chamber. Then, a total of 10<sup>9</sup> DRP of rAAV2/HBoV1 in 50 μL of medium were applied directly onto the apical surface of the airway epithelia at an MOI of ~2000 DRP/cell. At 12 hrs post-infection, the medium and virus were removed from the apical surface, and the basal medium was replaced with fresh medium without addition of proteasome inhibitors.

#### *2.9. Measurement of Luciferase Reporter Expression*

Luciferase enzyme activity was examined using a Luciferase Assay System kit (Promega, Madison, WI, USA) following the manufacturer's instructions. Briefly, HAE cells were collected after EDTA and trypsin treatments of the HAE-ALI cultures, and equal numbers of the cells from compared HAE-ALI cultures were transferred into wells of a 96-well plate. The wells were then added with 20 μL of 1× Lysis reagent, followed by mixing with 100 μL of Luciferase Assay Reagent and the light produced on a Synergy H1 microplate reader (Synergy H1, BioTek, Winooski, VT, USA) was measured.

#### *2.10. Antibodies Used*

A monoclonal antibody (clone 303.9) that reacts with AAV2 Rep78 and Rep52 and a monoclonal antibody (clone B1) that reacts with AAV2 Cap were purchased from American Research Products, Inc. (Waltham, MA, USA). Rat anti-HBoV1 Cap that reacts with VP1, VP2, and VP3 and rat anti-HBoV1 NP1 that recognizes the NP1 protein were made in house and have been described previously [26,31].

#### **3. Results**

#### *3.1. Design of the Baculovirus Expression vector System*

We used two BEVs, Bac-AAV2ITR-GFP and Bac-AAV2Rep-Cap (Figure 1E), to produce rAAV2 in Sf9 cells, which serves as a comparative control for the test of the production of the rAAV2/HBoV1 vector. In Bac-AAV2Rep-Cap, the AAV2 *rep* and *cap* genes were modified to allow expression of the Rep78 and Rep52 proteins and the VP1, VP2, and VP3 proteins from two species of mRNAs transcribed from Ph and P10 baculovirus promoters, respectively [56]. To generate the rAAV2/HBoV1 vector, we similarly made two BEVs, Bac-AAV2ITR-GFP-Luc, which harbors a 5.4-kb oversized rAAV2 genome, and Bac-AAV2Rep-HBoV1Cap (Figure 1A), which expresses AAV2 Rep78 and Rep52 under the Ph promoter and HBoV1 VP1, VP2 and VP3 under the P10 promoter. In addition, we made Bac-HBoV1NP1 that expresses the HBoV1 NP1 protein to look for a role of the NP1 in vector production.

We also compared the production and biologic properties of the rAAV2/HBoV1 vectors from insect cell and mammalian cell systems in parallel. To this end, we made the cis and trans plasmid constructs for rAAV2/HBoV1 vector production in HEK293 cells in the format similar to those used in the BEVs. The pAAV2ITR-GFP-Luc (5.4-kb) harbors the identical 5.4-kb rAAV2 genome that was in the transfer BEV Bac-AAV2ITR-GFP-Luc. The pCMVNS\*Cap-P5Rep is the HBoV1 *cap* and AAV2 *rep* two-in-one expression plasmid, which expresses HBoV1 NS (NP1, NS3 and NS4) and Cap (VP1, VP2, and VP3) under the cytomegalovirus immediate early promoter (CMV) [27,28] and AAV2 Rep78 and Rep 52 under the AAV2 P5 and P19 promoters, respectively (Figure 1B).

#### *3.2. Analyses of Protein Expression and Replication of the rAAV2 Genome in Sf9 Cells*

To characterize the expression of AAV Rep and Cap, Sf9 cells grown in suspension culture were infected with Bac-AAV2Rep-Cap, Bac-AAV2Rep-HBoV1Cap, and Bac-HBoV1NP1, respectively. The infected cells were collected at 72 hrs post-infection, and the expression of viral proteins was analyzed by Western blotting. We first examined the expression of AAV2 Rep from the Sf9 cells infected with Bac-AAV2Rep-Cap (Figure 2A) and Bac-AAV2Rep-HBoV1Cap (Figure 2B), respectively. The results of Western blotting showed that the expressions of the AAV2 Rep by the P10 promoter from these two BEVs demonstrated a similar pattern, which both expressed AAV2 Rep78 and Rep52 at a ratio close to ~1:2. Of note, when we constructed the Bacmid pBac-AAV2Rep-HBoV1Cap, the codon optimization of AAV2 rep (Figure 1C) was adopted from one mRNA transcript, which was used in Bac-AAV2Rep-Cap [56]. The HBoV1 Cap expression from the Sf9 cells infected with Bac-AAV2Rep-HBoV1Cap was also analyzed with Western blotting, which confirmed that the optimization of initiation codons (Figure 1D) led to the expression levels of HBoV1 VP1, VP2, and VP3 at a ratio close to ~1:1:10 (Figure 2C), similar to that was observed from the transfection of the pCMVNS\*Cap in HEK293 cells [28]. The cryptic polyadenylation signals (pA) resided inside the unique sequence in the VP1 ORF, which serve as the proximal pA preventing HBoV1 *cap* transcription in mammalian cells in the absence of NP1 expression [28], appeared to not be effective in the insect cells. These results confirmed that the expression strategy that Bac-AAV2Rep-Cap utilized to express the overlapping genes *rep* and *cap* from the baculoviral promoters P10 and Ph was applicable for the construction of Bac-AAV2Rep-HBoV1Cap. This AAV2/HBoV1 trans helper possessed the same capability to express AAV2 Rep proteins and also HBoV1 VP1, VP2 and VP3, and more importantly, they were expressed at the expected ratios. Thus, one BEV was able to express the parvoviral proteins from different genera efficiently without mutual disruption.

To determine the function of HBoV1 NP1 during the replication of the rAAV2 genome, we made a Bac-HBoV1NP1. It expressed HBoV1 NP1 at ~25 kDa (Figure 2D). Then, Sf9 cells were co-infected with Bac-AAV2ITR-GFP-Luc and Bac-AAV2Rep-HBoV1Cap, with or without Bac-HBoV1NP1. The infected cells were sampled at 72 hrs post-infection and analyzed for the presence of rAAV2 replicative-form (RF) DNA intermediates by Southern blotting (Figure 2E). Although ssDNA was not obviously detected in both groups, clearly much more double replicative form (dRF) DNA was observed in the presence of NP1 expression (Figure 2E, compare lanes 2 vs 3). Although NP1 is not required to modulate the HBoV1 cap expression in Sf9 cells as it does in mammalian cells, it positively enhances the replication of rAAV2 genomes. The mechanism of NP1's involvement in rAAV2 replication in Sf9 cells remains unclear.

**Figure 2.** Expression of AAV2 Rep and Cap proteins and HBoV1 Cap and NP1 proteins in BEV-infected Sf9 cells. (**A**–**D**) Western blotting. Sf9 insect cells were infected with Bac-AAV2Rep-Cap (**A**), Bac-AAV2Rep-HBoV1Cap (**B**,**C**), or Bac-HBoV1NP1 (**D**). The infected cells were collected at 72 hrs post-infection and subjected to Western blot analysis. (**A**) AAV2 Rep proteins were detected with an anti-Rep monoclonal antibody. (**B**,**C**) Bac-AAV2Rep-HBoV1Cap generated from transfection of three bacmid (Bacmid-1-3) were infected with Sf9 cells independently. (**B**) AAV2 Rep proteins were detected with an anti-Rep monoclonal antibody, and (**C**) HBoV1 Cap protein expression was detected with an anti-HBoV1 Cap protein antiserum. (**D**) HBoV1 NP1 was detected with a rat anti-HBoV1 NP1 antiserum. β-actin served as a loading control. Mock, uninfected cells. (**E**) Southern blotting. Sf9 cells were infected with Bac-AAV2ITR-GFP-Luc and Bac-AAV2Rep-HBoV1Cap with (+) or without (-) co-infection of Bac-HBoV1NP1. Cells were collected at 72 hrs post-infection and subjected to extraction of lower molecular weight (Hirt) DNAs, which were analyzed by Southern blotting. Mock, uninfected Sf9 cells as a control; M, a marker of a rAAV2ITR-GFP-Luc proviral replicative form (RF) genome of 5.4 kb. dRF and mRF, double and monomer RF.

#### *3.3. rAAV2*/*HBoV1 vector is Successfully Produced in Sf9 Cells and NP1 Plays a Role in Increasing vector Yield*

As a parallel control, we infected 200 mL of Sf9 cells with Bac-AAV2ITR-GFP and Bac-AAV2Rep-Cap for rAAV2 vector production. At 72 hrs post-infection, the infected cells were collected and lysed, and rAAV2 was purified by CsCl density gradient centrifugation. Fractions at a volume of 500 μL were collected and quantified for DRP by qPCR and demonstrated a peak at a refractive index of 1.372 (a density of ~1.40 g/mL) (Figure 3A, left). An electron micrograph of the rAAV2 produced is shown (Figure 3A, right) displaying particles of ~25 nm in diameter, a typical morphologic feature of AAV. In the peak fraction, the rAAV2 vector yield reached 7.52 <sup>×</sup> <sup>10</sup><sup>9</sup> DRP/μL, indicating that the Sf9 system to produce rAAV vector was successful at a yield of ~1 <sup>×</sup> 10<sup>4</sup> DRP/Sf9 cell from 200 mL Sf9 cells at a density of 2 <sup>×</sup> 106 cells/mL, a total 4 <sup>×</sup> 108 cells.

**Figure 3.** Purification of rAAV2 and rAAV2/HBoV1 vectors produced from BEV-infected Sf9 cells. (**A**–**C**) Vector production. Sf9 cells were co-infected with Bac-AAV2ITR-GFP and Bac-AAV2Rep-Cap (**A**), Bac-AAV2ITR-GFP-Luc and Bac-AAV2Rep-HBoV1Cap (**B**), or Bac-AAV2ITR-GFP-Luc, Bac-AAV2Rep-HBoV1Cap, and Bac-HBoV1NP1 (**C**). Cell lysates from infected cells were fractionated

by CsCl equilibrium ultracentrifugation. Left panel: qPCR analysis was used to determine the DRP in each fraction of 0.5 mL (blue line); the density of each fraction was determined as refractive index and is shown by the line in green. Right panel: transmission electron micrographs of rAAV2 or rAAV2/HBoV1 vectors, which were negatively stained with a 1% uranyl acetate solution. Bar = 100 nm. (**D**) Comparison of rAAV2/HBoV1 production with or without NP1 expression in Sf9 cells. The experiments in panels B&C were repeated three times in parallel. Purified vectors at the peak fraction were quantified and compared. Averages and standard deviations are shown. Statistical analysis was performed to get the P value using student "t" test.

The production of the rAAV2/HBoV1 vector was performed with BEV infection to Sf9 cells under the same conditions for rAAV2. We compared two groups of BEVs with or without expression of HBoV1 NP1 in parallel by infecting 4 <sup>×</sup> 108 cells of Sf9 cells: Group I, with Bac-AAV2ITR-GFP-Luc and Bac-AAV2Rep-HBoV1Cap; Group II, with Bac-rAAV2ITR-GFP-Luc, Bac-AAV2Rep-HBoV1Cap, and Bac-HBoV1NP1. At three days post-infection, the infected cells were collected and lysed, and vectors were purified by CsCl density gradient centrifugation. The refractive index and DRP of each fraction are shown at the left in Figure 3B,C, and the transmission electron microscopy demonstrated that the rAAV2/HBoV1 vector had a typical parvovirus icosahedral structure that was ~25 nm in diameter as shown at the right in Figure 3B,C. Without expression of HBoV1 NP1, an average vector yield was 1.6 <sup>×</sup> 109 DRP/μ<sup>L</sup> in the peak fraction of 500 <sup>μ</sup>L; however, there was a significant increase to 5.0 <sup>×</sup> 10<sup>9</sup> DRP/μL with the help of Bac-HBoV1NP1, confirming that expression of NP1 significantly increased vector yield by three times (Figure 3D). Notably, the expression of NP1 led to an increase in rAAV2 replicative-form (RF) DNA intermediates (Figure 2E), which could be responsible for the enhanced production.

#### *3.4. Comparison of the Transduction E*ffi*ciencies Between rAAV2*/*HBoV1 vectors Produced in Sf9 Cells and in HEK293 Cells*

It is encouraging that the yield of rAAV2/HBoV1 produced from the Sf9 cell system was comparable to that of rAAV2 in Sf9 cells in the presence of NP1 expression (5.0 <sup>×</sup> 10<sup>9</sup> vs 7.5 <sup>×</sup> 10<sup>9</sup> DRP/ul in the peak fraction of 500 μL). We next characterized its biological function in transducing HAE-ALI cultures. To this end and for fair comparison, we produced rAAV2/HBoV1(293) by transfection of pAAV2ITR-GFP-Luc, pCMVNS\*Cap-P5Rep and pHelper into HEK293 cells of 20 × 145-mm plates and obtained a yield of 2.3 <sup>×</sup> 109 DRP/μL at the peak fraction (Figure 4A). We apically infected the well-differentiated HAE-ALI cultures, which were generated from airway epithelial cells from two different donors, with equal amounts of vectors produced from Sf9 cells or HEK293 cells. Proteasome inhibitors LLnL and doxorubicin were only applied in the basal chamber during the infection period of 12 hrs [42]. At seven days post-infection, cells were examined for the GFP expression under a fluorescence microscope, and images were taken at the same setting (Figure 4B,D). We observed more green cells from the infection of rAAV2/HBoV1(293) with relatively stronger intensity of fluorescence than its counterpart infection transduced of the rAAV2/HBoV1(Sf9). Next, the cells were lysed for quantification of the luciferase activity (Figure 4C,E), which revealed that the rAAV2/HBoV1(293) vector has a transduction efficiency 5–7 times higher than the rAAV2/HBoV1(Sf9) vector.

**Figure 4.** Comparison of the transduction efficiency between the rAAV2/HBoV1 vectors produced in Sf9 cells and HEK293 cells. (**A**) rAAV2/HBoV1 vector produced from HEK293 cells. HEK293 cells were transfected with pAAV2ITR-GFP-Luc, pCMVNS\*Cap-P5Rep, and pHelper. Cell lysates from infected cells were fractionated by CsCl equilibrium ultracentrifugation. Left panel: qPCR analysis was used to determine the DRP in each fraction (blue line); the density of each fraction was determined as refractive index using an Abbe refractometer and is shown by the line in green. Right panel: a transmission electron micrograph of rAAV2/HBoV1(293) vectors. (**B**–**E**) rAAV2/HBoV1 transduction of HAE-ALI. HAE-ALI cultures prepared form Donor A (**B**,**C**) and Donor B (**D**,**E**) were transduced with rAAV2/HBoV1 either produced from Sf9 or HEK293 cells at an MOI of ~2000 DRP/cell from the apical surface. The rAAV2/HBoV1 vector was applied directly onto the apical surface of the airway epithelia. HAE cells were examined for GFP expression at 10 days post-transduction. Images were taken with an Eclipse Ti-S microscope (Nikon, Melville, NY, USA) at a magnification of × 20 (**B**&**D**). Luciferase activity was assayed at 10 days post-transduction (**C**&**E**). Averages and standard deviations generated from at least three independent experiments are shown. Statistical analysis was performed to get the P value using student "t" test.

#### **4. Discussion**

Cross-genera pseudopackaging between parvoviruses was first established by pseudotyping a rAAV genome into a capsid of human parvovirus B19 [61] for a chimeric AAV-B19 vector in HEK293 cells, which demonstrated high tropism to human erythroid cells. In 2013, we successfully packaged an rAAV2 genome into the capsid of HBoV1 in HEK293 cells, generating rAAV2/HBoV1 chimeric vector [42]. The rAAV2/HBoV1 vector has a high tropism for polarized human airway epithelia and is able to encapsidate an oversized rAAV2 genome of 5.8-kb, representing one of the best rAAV vectors for gene delivery to human airways and holding much promise for use in preclinical trials of CF gene therapy in ferrets and human trials of CF patients [62].

To meet the high demand of rAAV2/HBoV1 vector production at a large quantity, in this study, we took advantage of the rAAV2 vector production system in insect cells. We modified HBoV1 *cap* gene in Bac-AAV2Rep-HBoV1Cap that expressed VP1, VP2, and VP3 at a ratio of ~1:1:10 in Sf9 cells, and proved that the rAAV2/HBoV1 vector was produced in Sf9 cells. More importantly, with the co-infection of a BEV expressing HBoV1 NP1, the rAAV2/HBoV1 vector was produced at a yield of 5.0 <sup>×</sup> <sup>10</sup><sup>9</sup> DRP/μL, an equivalent efficiency as that of the rAAV2 vector in Sf9 cells (7.5 <sup>×</sup> 109 DRP/μL in the peak fraction) from a small suspension culture (200 mL of Sf9 cells at a density of 2 million/mL) (Table 1).


**Table 1.** Comparison of vector production in Sf9 vs HEK293 and with or without NP1 expression.

Note: 200 mL of Sf9 cells at a density of 2 × <sup>10</sup><sup>6</sup> cells/mL (a total of 4 × <sup>10</sup>8) and 20 145-mm plates of HEK293 cells (a total of 5 × 108) were infected /transfected for rAAV vector production. Vectors in the peak fraction that has the density of 1.40 g/mL in CsCl were quantified after dialyzed. \* Helper: other than *rep*/*cap* trans complementary.

The yield of rAAV2/HBoV1 in Sf9 cells is ~10–100-fold higher than in HEK293 cells, considering a yield per cell [47,53,54,56]. The infection of the BEVs to Sf9 cell suspension is simpler than the plasmid transfection to HEK293 cells, and the process is easily scalable for large preparation, e.g., with a Bio-Reactor. It was previously reported that the biological characteristics of Sf9 cell-produced rAAV is equivalent to the HEK293 cell-produced rAAV [47,48,53,63]. However, in contrast, we observed that the transduction activity of the rAAV2/HBoV1(Sf9) vector produced from Sf9 cells is 5–7 times lower than that of the rAAV2/HBoV1(293) vector packaged in HEK293 cells (Figure 4C,E). We speculated that the rAAV2 genomes may not be as well packaged in HBoV1 capsids as that in AAV2 capsids, thus we examined these vector preps under transmission electron microscopy. We noticed that the rAAV2/HBoV1(293) vector barely had any empty particles (>95% full particles) (Figure 4A) as did the rAAV2 vector produced from Sf9 cells (Figure 3A, right panel), whereas rAAV2/HBoV1(Sf9) vectors had a high level of empty particles (only 50–60% full particles) (Figure 3B,C, right panels). Infection of BEV-Rep2Cap2, which was made following the Kotin strategy of Bac-AAV2Rep-Cap [56], expressed AAP in Sf9 cells, and knockout of the AAP decreased rAAV2 yield by 10 times [55]. This suggested that the AAP plays an important role in rAAV vector production in Sf9, which is likely through facilitation of the assembly of AAV capsids [22–24]. We currently do not know whether HBoV1 *cap* also expresses an AAP-like protein that may facilitate the assembly of the HBoV1 capsid, which warrants further investigation. Recently, glycosylation of rAAV has been reported and likely affects the potency of vector [64]. The possible variations in glycosylation between the vectors produced in HEK293 and Sf9 cells may also impact the transduction.

While the replication of the rAAV2 genome in Sf9 is not the rate limiting step for both rAAV2 and rAAV2/HBoV1 productions, it appears the trans functions for the pseudopackage of the rAAV2 genome in HBoV1 are less efficient than that for packaging it in the capsid of AAV2 or another AAV serotype. We have demonstrated an HBoV1 NS-free production system for rAAV2/HBoV1 in HEK293 cells [45]. In such a case, it appears that the expressions of AAV Rep proteins together with the helper components of adenovirus are sufficient for the cross-genera pseudopackage. It is clear that the adenovirus helper

functions are not essential to the production of rAAV2 in Sf9 cells; however, it remains unknown whether they play a role in assisting the package of the rAAV2 genome into the HBoV1 capsid in HEK293 cells. Of note, the helper components from adenovirus are absent in the BEV system. Thus, the AAV2 Rep proteins, especially the AAV2 Rep52, might not be acting as efficiently in Sf9 cells as it does for pseudopackage of the rAAV2 genome in the HBoV1 capsid in HEK293 cells. For future improvement, we will tackle whether incorporation of one or more adenovirus helper components will solve this problem. Another consideration is the potent involvement of HBoV1 small NS proteins, despite the fact that the NS-free rAAV2/HBoV1 vector production system in HEK293 cells is against such action [45]. However, it is possible that they might confer necessary function in the absence of adenovirus function. Among them, the NS3 might be the first choice for the test, as it fully contains the helicase domain of the NS1, which is similar to the AAV2 Rep52 in structure [27] and executes helicase activity during viral genome packaging as the AAV2 Rep52 does [65].

In conclusion, we have established a rAAV2/HBoV1 vector production system in suspension culture of Sf9 cells for pseudopackage of the rAAV2 genome into the HBoV1 capsid. The yield of the rAAV2/HBoV1 vector is similar to that of rAAV2 produced in suspension Sf9 culture in a small volume, which is scalable in a large culture of Sf9 cells [49–51,53,66,67]. However, the current rAAV2/HBoV1-BEV system tends to produce more empty particles than the counterpart rAAV2 vector system. In the future, we will optimize the Sf9 cell production and purification system to reduce empty particles and to produce the rAAV2/HBoV1 vector in a large quantity as the suspension Sf9 cell culture can be easily scaled, which will enable the use of the vector for gene therapy of CF lung disease in large animal models.

**Author Contributions:** Conceptualization, Z.Y. and J.Q.; methodology, X.D.; validation, X.D.; formal analysis, X.D.; investigation, X.D.; resources, X.D. and W.Z.; data curation, X.D.; writing—original draft preparation, X.D. and J.Q.; writing—review and editing, X.D., W.Z., Z.Y., and J.Q.; visualization, X.D.; supervision, J.Q.; project administration, J.Q.; funding acquisition, Z.Y. and J.Q. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by PHS grants AI150877 and AI139572 from the National Institute of Allergy and Infectious Diseases. This study was also supported by grant YAN19XX0 from the Cystic Fibrosis Foundation. The Electron Microscope Research Laboratory is supported, in part, by NIH/NIGMS COBRE grant P20GM104936. The JEOL JEM-1400 transmission electron microscope was purchased with funds from NIH grant 1S10RR027564. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

**Acknowledgments:** We thank the members of the Qiu lab for technical support and valuable discussions. We acknowledge the Electron Microscope Research Laboratory, The University of Kansas Medical Center, for help with transmission of electron microscopy.

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

#### **References**


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