**6. Challenges to pulmonary gene transfer with integrating vectors**

### **6.1. Delivery**

determine the preferred gene transfer targets and the level of CFTR correction required to

In pre-clinical studies of CF gene therapy, it is vital to define metrics of correction before the studies are initiated. It would be naïve to simply deliver vector to the airways and "look and see" if the airway disease is cured. CF is a complex disease with many phenotypic features and clearly defining the disease progression in an untreated CF animal model is vital. As experience is gained with new animal models, additional assays for correction will be established and refined. Importantly, these metrics may apply to multiple gene correction but may not be

Quantitative real-time RT-PCR is a sensitive assay for measuring vector expressed *CFTR* mRNA [123]. At progressive time-points post vector delivery, whole tissue or brushings of nasal or tracheobronchial epithelia can be obtained. Silent mutations can be engineered into the vector expressed *CFTR* cDNA so that transgene expression can be differentiated from endogenous *CFTR*. Using a similar strategy, vector genome copy number can be estimated. Genomic DNA from a portion of the same tissue or epithelial brushings can be purified and copy number estimated by normalizing to endogenous DNA and a standard curve. Appro‐ priate controls would include wild type and untreated affected littermates. To identify cells expressing the CFTR protein, immunohistochemistry and immunofluorescence protocols have been reported [123]. Using these approaches, the percentage of cells expressing CFTR and the

Nasal potential difference (NPD) is an established assay for demonstrating *in vivo* correction

nasally and the NPD was used as evidence of CFTR complementation. Ideally, for integrating vectors, predelivery, early post-delivery, and late post-delivery timepoints in the intrapulmo‐ nary airways should be measured. As early as 1 week after gene transfer, the nasal voltage and

could be followed with serial monthly nasal voltage measurements over a 12-month or longer period to document persistence of expression. Importantly, CFTR is an anion channel that

benchmark for restoring Cl- transport and correcting the clinical phenotype. However, other

studies of CF mouse cervical mucus [46, 47], CF mouse small intestinal mucus [48], and human


CF nasal SMGs [49] support the importance of CFTR-dependent HCO3

transport [125-127]. For many of these studies, vector was delivered

, and cAMP agonists can be measured [90, 123, 128]. Animals

transport might be relevant to disease. Multiple



transport in CF

. As mentioned above, correcting ~10% of cells is often cited as a

prevent or slow disease progression

**5. Outcome measures**

336 Cystic Fibrosis in the Light of New Research

feasible in all animal models.

**5.2. Functional correction**

of CFTR-dependent Cl-

conducts both Cl-

its response to amiloride, low Cl-

and HCO3

pathogenesis. Loss of CFTR-dependent HCO3

studies also suggest that defective HCO3


**5.1. Quantitative real-time PCR and CFTR protein expression**

cell types expressing CFTR can be determined [124].

The lung is an attractive target for gene therapy because, unlike most other tissues, the vector can be topically delivered. Vector delivery to the airways of mice, rats, and newborn ferrets is most easily accomplished by nasal or intratracheal bolus delivery of vector resuspended in a liquid vehicle such as buffered saline [135], LPC [60, 62], or a viscoelastic gel [68, 69]. In small animals, bolus delivery using a relatively small volume of vector (25-50 μl) can achieve widespread gene expression throughout the airways. However, in large animal models such as pigs or sheep (and ultimately humans), aerosolization will likely be required to achieve a widespread pulmonary distribution. In general, devices for generating airborne vector fall into three categories: aerosolizing catheters, nebulizers, and atomizers. All of these devices convert liquids into particles small enough to be respired.

Aerosolizing catheters convert liquids into particles at the point of expulsion. Typically, an aerosolizing catheter is first passed into the trachea and then the vector is instilled. The Microsprayer® (PennCentury) and the AeroProbe® catheter (Trudell Medical International) are examples of delivery systems for this application. According to the manufacturer, it is possible to generate particles with aerodynamic diameters of 4–8 μm. The Trudell AeroProbe was previously used to aerosolize helper-dependent adenovirus vectors to rabbit airways [136, 137] and to deliver Sendai virus vectors to sheep [138]. Since Sendai virus and lentiviruses are both enveloped, it is likely that this approach is feasible with lentiviral vectors. In addition, aerosolized VSVG-LVs have been successfully delivered to the airways of mice [139].

Successful vector aerosolization has been reported in mice [140], rabbits,[136, 137], pig [141] and sheep airways [142, 143]. By 2–3 weeks of age, wild type pigs are large enough to be sedated and have a pediatric bronchoscope passed into the trachea. The AeroProbe® catheter can be passed to the carina via the bronchoscope suction channel with the animal breathing sponta‐ neously. In this way, an integrating vector can be aerosolized and targeted to specific bronchial segments.

In the case of nebulizers, the liquid is first converted into mist and then passively inhaled. Using this strategy, a plasmid-based vector was delivered to the airways of CF patients in a phase IIB gene therapy trial [144]. This approach could potentially be used to deliver DNA transposon or hybrid vectors. However, this strategy may not be feasible with enveloped viral vectors because this class of vectors may not be stable enough to withstand nebulization. In addition, nebulization requires a large volume of concentrated material; therefore, would be the least economic delivery strategy for LVs.

Atomizers are a subclass of aerosolizing catheters that deliver larger sized particles. An atomizer, such as the MADgicTM (LMA) atomizer, delivers large droplets (~30–90 μm in diameter), which may vary in size depending on the force applied to the syringe plunger. This type of device is often used to topically deliver medications to the airways [145]. Our group has observed that this type of atomizer is an effective delivery device for multiple viral vectors including FIV, particularly when formulated with a viscoelastic material such as methylcel‐ lulose.

### **6.2. Insertional mutagenesis**

Since persistent gene expression from lentiviral vectors requires genomic integration, they show promise for treating life-long genetic diseases; however, there is inherent risk when introducing a transgene with integrating vectors. Insertional mutagenesis may disrupt normal cell functions by inactivating an essential host gene or inappropriately causing expression of an undesirable gene. The risk will vary depending on the vector used, the transgene cassette, and the cell type targeted. In many cases, enhancer effects pose the greatest danger. So far, malignant cell transformation after vector-mediated insertional mutagenesis has only been observed in three clinical entities (X-linked severe combined immunodeficiency (SCID-X1), chronic granulomatous disease (CGD), and Wiskott–Aldrich syndrome (WAS)), all of which occurred in conjunction with the use of first-generation gamma-retroviral vectors harboring LTRs with strong enhancer/promoter sequences [146-150]. These studies were conducted in immunocompromised patients where gene transfer conferred a selective advantage to corrected cells. The vector and disease settings likely influenced the risks for insertional mutagenesis and subsequent clonal expansion. Modern LVs are engineered to lack enhancer/ promoter sequences within the LTRs and delivering *CFTR* to somatic cells has no known selective advantage. Considerable effort has been put toward mapping integration patterns and determining the functional consequences of LVs and retroviral vectors (reviewed in [151, 152]). LV integration analyses conducted on adrenoleukodystrophy clinical trial patients demonstrate that the genomic distribution maintain a polyclonal pattern [153]. Montini and colleagues demonstrated that LV integrations, even at high vector titer loads, did not accelerate tumorigenesis in tumor prone mice. In contrast, gamma-retroviral vector transduction triggered a dose-dependent acceleration of tumor onset [154].

The burden of proof has fallen on LV researchers to demonstrate that LVs do not cause cancer via insertional mutagenesis. It is unlikely that this can ever be demonstrated with absolute certainty; however, the evidence to date suggests that current LVs are considera‐ bly safer than the gamma-retroviral vectors that were first brought to clinical trials. In fact, results from human clinical trials using LVs are encouraging and the feasibility of gene therapy for monogenetic diseases is now firmly established [155]. Recent promising examples include Wiskott–Aldrich Syndrome [156, 157], metachromatic leukodystrophy [158, 159], acute lymphoid leukemia [160], lymphoma [161, 162], and multiple primary immuno-deficiencies [163].
