**3. Options for integrating gene delivery**

### **3.1. Lentivirus**

Lentiviruses comprise a genus of the virus family *Retroviridae.* All retroviruses are defined by the ability to reverse transcribe their RNA genome and integrate proviral DNA into the genome of the host cell [50]. Several features of lentiviral vectors (LVs) make them attractive vehicles for delivering therapeutic genes, including their large coding capacity, efficient gene transfer, persistent expression, directed tropism via pseudotyping, and lack of virus-encoded proteins that could elicit undesirable immune responses [51-53]. Unlike gamma-retroviral vectors such as murine Moloney leukemia virus (MMLV)-based vectors, the pre-integration complex of lentiviruses can transverse the nuclear envelope and integrate its cargo into the genomes of non-dividing cells [54]. The first recombinant lentiviral vectors were based on human immu‐ nodeficiency virus type-1 (HIV-1) and remain the most widely used lentiviral vector for gene transfer applications [55]. The earliest recombinant HIV-1 viruses were created in the late 1980s and were used to study HIV-1 biology [56].

Beginning in the 1990s and continuing to this day, there have been significant strides in improving the safety and utility of HIV-based gene transfer vectors (reviewed in [50]). To reduce pathogenicity and render the vector replication incompetent, most modern HIV production systems are divided into four expression plasmids (Figure 1A): 1) a plasmid containing the transgene of interest flanked by the HIV long terminal repeats (LTRs); 2) a packaging plasmid expressing the necessary structural and enzymatic proteins; 3) a separate plasmid expressing HIV rev; and 4) an envelope glycoprotein expression plasmid. Multi‐ ple viral proteins, such as nef, vif, vpu, env, and vpr, have been deleted as well as much of the U3 region of the 3'LTR. This latter deletion removes promoter and enhancer activity from the LTR and has been termed a self-inactivating (SIN) modified vector [57]. The woodchuck hepatitis virus posttranscriptional regulatory element (wPRE) is a common addition to LV vectors and functions to increase RNA stability, resulting in higher levels of transgene expression [58, 59].

Integrating Viral and Nonviral Vectors for Cystic Fibrosis Gene Therapy in the Airways http://dx.doi.org/10.5772/60977 331

HCO3 -

vectors are being engineered.

330 Cystic Fibrosis in the Light of New Research

**3.1. Lentivirus**

Cl-

transport through CFTR might be more relevant to early disease pathogenesis than

bacterial killing, mucociliary clearance, and mucus viscosity may be as important as Clsecretion as metrics for disease correction. The short answer to the question posed above is "we do not know"; however, given current animal models and improved vector technologies, the experiments necessary to address the question are feasible. Indeed, as we discuss below, existing vector technologies are being optimized for lung gene transfer and novel integrating

Lentiviruses comprise a genus of the virus family *Retroviridae.* All retroviruses are defined by the ability to reverse transcribe their RNA genome and integrate proviral DNA into the genome of the host cell [50]. Several features of lentiviral vectors (LVs) make them attractive vehicles for delivering therapeutic genes, including their large coding capacity, efficient gene transfer, persistent expression, directed tropism via pseudotyping, and lack of virus-encoded proteins that could elicit undesirable immune responses [51-53]. Unlike gamma-retroviral vectors such as murine Moloney leukemia virus (MMLV)-based vectors, the pre-integration complex of lentiviruses can transverse the nuclear envelope and integrate its cargo into the genomes of non-dividing cells [54]. The first recombinant lentiviral vectors were based on human immu‐ nodeficiency virus type-1 (HIV-1) and remain the most widely used lentiviral vector for gene transfer applications [55]. The earliest recombinant HIV-1 viruses were created in the late 1980s

Beginning in the 1990s and continuing to this day, there have been significant strides in improving the safety and utility of HIV-based gene transfer vectors (reviewed in [50]). To reduce pathogenicity and render the vector replication incompetent, most modern HIV production systems are divided into four expression plasmids (Figure 1A): 1) a plasmid containing the transgene of interest flanked by the HIV long terminal repeats (LTRs); 2) a packaging plasmid expressing the necessary structural and enzymatic proteins; 3) a separate plasmid expressing HIV rev; and 4) an envelope glycoprotein expression plasmid. Multi‐ ple viral proteins, such as nef, vif, vpu, env, and vpr, have been deleted as well as much of the U3 region of the 3'LTR. This latter deletion removes promoter and enhancer activity from the LTR and has been termed a self-inactivating (SIN) modified vector [57]. The woodchuck hepatitis virus posttranscriptional regulatory element (wPRE) is a common addition to LV vectors and functions to increase RNA stability, resulting in higher levels


secretion, airway surface liquid pH,

secretion [46-49]. The relationships between HCO3

**3. Options for integrating gene delivery**

and were used to study HIV-1 biology [56].

of transgene expression [58, 59].

**Figure 1.** Schematics of integrating transgene delivery systems. A) HIV-based lentiviral vectors are produced by four plasmid transfection. The gag/pol plasmid supplies structural and enzymatic proteins; the env plasmid supplies the envelope glycoprotein (typically VSV-G); rev is delivered *in trans* via its own plasmid. The "gene of interest" (goi) is flanked by the long terminal repeats (LTRs) and driven by a heterologous promoter. Only the genetic material flanked by the LTRs is packaged and integrated into the host genome. B) Recombinant DNA transposons are typically deliv‐ ered as a two-part plasmid system. Transposase catalyzes the transposition of the genetic material flanked by the ap‐ propriate terminal repeats (TRs) from the plasmid and into the host genome. C) DNA transposons can also be delivered by viral vectors to improve delivery efficiency. One viral vector carries the DNA transposon and the other carries the transposase. The goi is flanked by the transposon TRs, which in turn are flanked by the viral vector inverted terminal repeats (ITRs). Once inside the cell, transposition functions as described in B.

HIV-based LV delivery of CFTR is a promising option for CF gene therapy. In proof of principle experiments using reporter genes, HIV conferred gene transfer to both ciliated and basal cells of the mouse, sheep, marmoset, and ferret airways [60]. Xenografts transduced with HIV expressing CFTR achieved functional correction as assessed by the measurement of transepi‐ thelial potential difference [61]. In a CF mouse model, delivery of CFTR by HIV resulted in sustainable transgene expression for 18 months in ciliated, non-ciliated, and basal cells [53] and partially recovered the anion channel defect [62]. These findings support HIV as a vector candidate for CF gene therapy. Further studies in a large animal CF model to restore anion channel defect to rescue pH and bacterial killing are important next steps in validating this vector for CF gene therapy.

For LVs, the native envelope glycoprotein is deleted and a heterologous envelope is supplied. This strategy, termed pseudotyping, modifies vector tropism. Glycoproteins from a wide variety of enveloped viruses can be used to package LVs and, as will be discussed, multiple groups have identified envelopes that confer lung gene transfer. The envelope glycoprotein from vesicular stomatitis virus (VSV-G) efficiently pseudotypes LVs, confers wide tropism, and is the most commonly used envelope glycoprotein. However, in well-differentiated airway epithelial cells, VSV-G pseudotyped LV (VSVG-LV) preferentially transduces the basolateral surface [63, 64]. By using pretreatments or vehicles that transiently disrupt epithelial tight junctions, VSVG-LV accesses the basolateral surface of airway cells following luminal delivery [65]. This strategy has also been shown to greatly improve VSVG-LV *in vivo* gene transfer efficiency in the lungs of mice [62].

Feline immunodeficiency virus (FIV) is a non-primate LV with a less complex genome than HIV. Unlike HIV, wild-type FIV naturally lacks tat, vpr, vpu, and nef. In addition, FIV-based vector has vif deleted. Additional modifications to the FIV vector, such as deleting the major splice donor, lead to a higher transduction efficiency and transgene expression [58]. Reporter gene studies demonstrate that FIV transduces cells in the conducting airways, bronchioles, and alveoli [66]. FIV-mediated delivery of CFTR corrects the anion channel defect in airway epithelia [65]. FIV pseudotyped with the baculovirus envelope glycoprotein (GP64) preferen‐ tially transduces polarized airway epithelia at the apical surface [63], results in persistent gene expression in mice [63], and supports gene transfer to airways of pigs [66]. Additionally, FIV vectors can be readministered to mouse airways without blocking immune responses [67]. To prolong virus exposure to the airways immediately following delivery, formulating vector with a viscoelastic gel increases transduction efficiency *in vivo* [68, 69].

Simian immunodeficiency virus (SIV)-based LV also successfully transduces airway epithelia. SIV carries little pathogenicity for its own host; therefore, modified strains of SIV could potentially be a safer alternative to HIV-based LVs [70]. In studies using Sendai virus envelope proteins (F and HN) pseudotyped SIV (F/HN-SIV), a single dose persisted for the lifetime in the nasal epithelia of a mouse and achieved a dose-dependent increase in reporter gene expression upon vector readministration [71]. *In vitro*, F/HN-SIV carrying CFTR can generate functional chloride channels [72]. Additionally, SIV transduction results in persistent trans‐ gene expression in both differentiated human airway cells and freshly excised human lung tissue [71].

Equine Infectious Anemia Virus (EIAV) is another non-primate lentivirus that has been investigated as a gene transfer vector. It has been studied extensively for neurological disease applications, such as Parkinson's disease [73]. EIAV pseudotyped with the influenza HA envelope can transduce neonatal mouse airways, most notably, the nasal and lung epithelium. Readministration of HA-EIAV resulted in decreased gene transfer efficiency [74].

### **3.2. DNA transposons**

HIV-based LV delivery of CFTR is a promising option for CF gene therapy. In proof of principle experiments using reporter genes, HIV conferred gene transfer to both ciliated and basal cells of the mouse, sheep, marmoset, and ferret airways [60]. Xenografts transduced with HIV expressing CFTR achieved functional correction as assessed by the measurement of transepi‐ thelial potential difference [61]. In a CF mouse model, delivery of CFTR by HIV resulted in sustainable transgene expression for 18 months in ciliated, non-ciliated, and basal cells [53] and partially recovered the anion channel defect [62]. These findings support HIV as a vector candidate for CF gene therapy. Further studies in a large animal CF model to restore anion channel defect to rescue pH and bacterial killing are important next steps in validating this

For LVs, the native envelope glycoprotein is deleted and a heterologous envelope is supplied. This strategy, termed pseudotyping, modifies vector tropism. Glycoproteins from a wide variety of enveloped viruses can be used to package LVs and, as will be discussed, multiple groups have identified envelopes that confer lung gene transfer. The envelope glycoprotein from vesicular stomatitis virus (VSV-G) efficiently pseudotypes LVs, confers wide tropism, and is the most commonly used envelope glycoprotein. However, in well-differentiated airway epithelial cells, VSV-G pseudotyped LV (VSVG-LV) preferentially transduces the basolateral surface [63, 64]. By using pretreatments or vehicles that transiently disrupt epithelial tight junctions, VSVG-LV accesses the basolateral surface of airway cells following luminal delivery [65]. This strategy has also been shown to greatly improve VSVG-LV *in*

Feline immunodeficiency virus (FIV) is a non-primate LV with a less complex genome than HIV. Unlike HIV, wild-type FIV naturally lacks tat, vpr, vpu, and nef. In addition, FIV-based vector has vif deleted. Additional modifications to the FIV vector, such as deleting the major splice donor, lead to a higher transduction efficiency and transgene expression [58]. Reporter gene studies demonstrate that FIV transduces cells in the conducting airways, bronchioles, and alveoli [66]. FIV-mediated delivery of CFTR corrects the anion channel defect in airway epithelia [65]. FIV pseudotyped with the baculovirus envelope glycoprotein (GP64) preferen‐ tially transduces polarized airway epithelia at the apical surface [63], results in persistent gene expression in mice [63], and supports gene transfer to airways of pigs [66]. Additionally, FIV vectors can be readministered to mouse airways without blocking immune responses [67]. To prolong virus exposure to the airways immediately following delivery, formulating vector

Simian immunodeficiency virus (SIV)-based LV also successfully transduces airway epithelia. SIV carries little pathogenicity for its own host; therefore, modified strains of SIV could potentially be a safer alternative to HIV-based LVs [70]. In studies using Sendai virus envelope proteins (F and HN) pseudotyped SIV (F/HN-SIV), a single dose persisted for the lifetime in the nasal epithelia of a mouse and achieved a dose-dependent increase in reporter gene expression upon vector readministration [71]. *In vitro*, F/HN-SIV carrying CFTR can generate functional chloride channels [72]. Additionally, SIV transduction results in persistent trans‐ gene expression in both differentiated human airway cells and freshly excised human lung

vector for CF gene therapy.

332 Cystic Fibrosis in the Light of New Research

tissue [71].

*vivo* gene transfer efficiency in the lungs of mice [62].

with a viscoelastic gel increases transduction efficiency *in vivo* [68, 69].

Recombinant DNA transposons are integrating nonviral vectors that confer efficient and stable transgene expression in a variety of cell types. The DNA transposon *Sleeping Beauty* (SB) was resurrected from defective copies of a Tc1-like fish element. SB transposes its genetic cargo into host genomic loci using its catalytic transposase activity [75]. Recombinant DNA trans‐ posons used for gene transfer applications are comprised of a two-part system; one encodes terminal repeats (TRs) flanking a transgene of interest (transposon), and the other a catalytic protein responsible for transposition (transposase) (Figure 1B). Since the discovery of SB, other transposons such as *piggyBac* and Tol2 have also been used in gene transfer applications (reviewed in [76, 77]). DNA transposons are attractive tools for gene therapy because they have a large carrying capacity and integrate into the genome.

Generally, transposon-based vectors are delivered as plasmids. This poses limitations for delivery to some somatic cell types. To improve delivery, several formulations have been investigated. Belur and colleagues described a protocol for complexing SB with polyethyle‐ neimine (PEI) for delivery to the airways of mice [78]. The efficiency of gene transfer was approximated to be 1%–3% 2 months post delivery. The use of liposomes has also been investigated as an alternative transposon delivery strategy. Somatic delivery of liposome– protamine formulated SB resulted in a higher transduction efficiency than Lipofectamine 2000 while maintaining low toxicity and biocompatibility [79]. This non-viral vector approach overcomes biological barriers and allows for chromosomal integration; however, to date, this strategy has not been applied to delivering CFTR to the *in vivo* airways.

### **3.3. Hybrid DNA transposon/viral vectors**

Delivery of plasmid-based transposon vectors to somatic cells *in vivo* is inefficient. To overcome this limitation, multiple groups have used viral vectors to deliver transposon components (Figure 1C). Recombinant DNA transposons delivered by non-integrating viral vectors, termed *hybrid vectors*, are intended to combine the advantage of efficient transduction of the viral vector with persistent expression of the transposon, creating an integrating vector. Adenovirus (Ad) [80, 81], adeno-associated virus (AAV) [82], and integrase-deficient lentivirus (ID-LV) [83, 84] have all been investigated as delivery tools for DNA transposons. An inte‐ gration analysis of SB delivered by ID-HIV suggests a similar, near random, pattern as is observed for plasmid-delivered SB [83].

Yant et al. showed that SB delivered by a helper-dependent Ad vector can integrate into the host chromosome in a transposase-dependent mechanism [75, 80]. Interestingly, in this system, the initial transposition of SB out of the Ad genome required the use of Flp-recombinase to successfully deliver the transposon transgene [80]. More recently, the generation of a hybrid *piggyBac*/Ad and *piggyBac*/AAV facilitated delivery of a nonviral transposon. However, unlike SB, neither *piggyBac*/Ad nor *piggyBac*/AAV required an extra recombinase step for transposi‐ tion to occur [81]. In contrast to the hybrid SB*, piggyBac* has not exhibited overexpression inhibition or limitations on size of the genetic cargo [85]. *PiggyBac*/Ad hybrid vector success‐ fully delivered a CFTR expression cassette to primary airway epithelial cultures *in vitro* that corrected the anion transport defect up to 4 months in culture. In reporter gene studies, transgene expression persisted for the 1-year duration of the experiment in mice [81].
