**Gene therapy: Gene supplementation and transcript supplementation therapy**

Instead of addressing the functional deficit in a patient's endogenous CFTR, another approach involves supplementing the cell with an exogenous, functional copy of the protein (Figure 2). This can occur in one of several ways: through gene, transcript, or protein replacement therapy. By delivering a functional copy of the *CFTR*, subsequent mRNA transcript, or protein itself, the cell may regain enough CFTR function to halt the progression of disease. Protein replace‐ ment strategies have met limited success, as the therapeutic protein is often metabolized before it can enter the target tissue. Gene and transcript therapy approaches, however, continue to be investigated. Unlike CFTR modulators, these approaches have the potential to be used for all CF patients, regardless of the type of mutation they carry.

**Figure 2. Gene, transcript, and protein replacement therapy supplement cells with a functional copy of the CFTR protein.** Supplementing the cell with functional *CFTR* cDNA **(A)**, mRNA transcripts **(B)**, or CFTR protein **(C)** is anoth‐ er method of overcoming the genetic defects underlying Cystic Fibrosis.

### **4.1. Gene supplementation therapy**

a non-conventional mode of gating, ivacaftor increases the probability that the chloride channel is open [14]. Another small molecule CFTR modulator, ataluren (PTC124, PTC Therapeutics, trade name Translarna in the EU), is a production corrector that makes ribo‐ somes less sensitive to G542X or W1282X nonsense mutations [15]. Overcoming these prema‐ ture stop codons allows the synthesis of full-length, functional CFTR. Together, these small molecule modulators focus on addressing the functional defects in a patient's own CFTR

**Figure 1. Small molecule CFTR modulators target functional defects within the CFTR protein.** Three types of CFTR modulators have been developed: premature stop codon suppressors **(A)**, correctors **(B)**, and potentiators **(C)**. These small molecules act by targeting the transcription, translation, protein processing, membrane trafficking, and ion trans‐

**Gene therapy: Gene supplementation and transcript supplementation**

Instead of addressing the functional deficit in a patient's endogenous CFTR, another approach involves supplementing the cell with an exogenous, functional copy of the protein (Figure 2). This can occur in one of several ways: through gene, transcript, or protein replacement therapy. By delivering a functional copy of the *CFTR*, subsequent mRNA transcript, or protein itself, the cell may regain enough CFTR function to halt the progression of disease. Protein replace‐ ment strategies have met limited success, as the therapeutic protein is often metabolized before it can enter the target tissue. Gene and transcript therapy approaches, however, continue to

protein.

port functionality of the CFTR protein, respectively.

360 Cystic Fibrosis in the Light of New Research

**therapy**

The two main forms of gene supplementation therapy are defined by the vehicle used to deliver functional cDNA to the cell: this consists of either viral or non-viral vectors (Figure 2A).

**Non-viral vectors** are typically comprised of plasmid DNA (pDNA) complexed with carrier molecules, such as cationic lipids or polymers. By binding to the negatively charged pDNA, these molecules either condense or encapsulate the DNA, forming lipoplexes or polyplexes that are then thought to be endocytosed by the cell [16]. In the absence of non-human, viral protein components, it is believed that non-viral vectors may incite minimal immune activation and increase the opportunity for repeat administration. However, even pDNA expression is often limited by CpG motifs that induce strong immune responses through innate immune receptors, such as Toll-like receptor 9 (TLR9) [17]. In addition, non-viral vectors are typically much less efficient than viral vectors at transfecting slowly dividing mammalian cells. This is due to the fact that viruses have evolved efficient strategies for improving cell entry, endosomal escape, cytoplasmic trafficking, and nuclear uptake, all of which make them naturally skilled vehicles for delivering therapeutic cDNA to the cell nucleus [16].

**Viral vectors** have been designed to harness these evolutionary advantages, while removing components of the viral genome that may cause harm. The ideal viral vector should be replication defective, non-immunogenic, and avoid integrating into actively transcribed genes. Random integration events into an oncogene or tumor suppressor may cause insertional mutagenesis leading to cell death or cancer.

**Adenoviral vectors (Ad)** engineered to be devoid of the viral genome were the first to be utilized for CF gene supplementation therapy. These vectors have the advantage of being nonintegrating, with a natural tropism for the lung. In clinical trials using Ad-CFTR, low levels of gene transfer and partial correction of chloride transport in nasal epithelium were observed in some patients [16]. However, issues such as dose-dependent lung inflammation and humoral and cellular immune responses preventing repeat administration remained limiting factors.

**Adeno-associated viral vectors (AAV)** also remain largely episomal inside the nucleus, minimizing the threat of insertional mutagenesis. Over 130 serotypes of AAV have been identified, with each viral capsid demonstrating its own unique transduction profile [18]. Capsids from AAV1, 5, 6, 8, and 9 may be the most efficient for transducing cells of the airway epithelium [19]. In addition, the creation of hybrid AAV capsids, such as AAV6.2, may allow the customization of vectors optimized for transducing the desired target cell. Early phase I trials with AAV2.CFTR showed limited efficacy, due in part to the use of a non-lung-tropic AAV2 serotype, limited packaging space for an optimal promoter (*CFTR* cDNA uses 4.7kb of the vector's ~5kb packaging capacity), as well as AAV capsid-specific immune responses limiting repeat administration [16]. Strategies aimed at minimizing adaptive immunity to AAV vectors or reducing the need for repeat administration continue within the field. Removing CpG motifs from AAV vectors or designing hybrid AAV capsids has been shown to reduce innate and adaptive immune responses following intramuscular delivery [20,21]. Targeting AAV delivery to progenitor cells in mouse lung also shows promise as a means of avoiding lung cell turnover and circumventing the need for redelivery [22].

**Lentiviral vectors** based on recombinant human (HIV), simian (SIV), feline (FIV), and equine (EIV) immunodeficiency viruses have also been investigated for gene replacement therapy [16]. Lentiviral vectors are pseudotyped with the envelope proteins from various viruses to increase tissue tropism. The vesicular stomatitis virus G (VSV-G) envelope glycoprotein has most commonly been incorporated, although the F and HN proteins from murine parain‐ fluenza virus type 1, or Sendai virus (SeV), may improve airway transduction. Studies with SeV-pseudotyped lentiviral vectors have accommodated repeat administration to murine airways in pre-clinical studies [23]. Should repeat administration also be feasible in human subjects, the two major remaining limitations to lentiviral use include safety concerns over genomic integration and scale-up of vector production. The concern over vector integration came to the forefront in 2003, when the integration of a retroviral vector used to treat X-linked severe combined immunodeficiency (X-SCID) triggered unexpected activation of a protooncogene leading to leukemia in nearly half of the trial's participants [24,25].

### **4.2. Transcript supplementation therapy**

**Viral vectors** have been designed to harness these evolutionary advantages, while removing components of the viral genome that may cause harm. The ideal viral vector should be replication defective, non-immunogenic, and avoid integrating into actively transcribed genes. Random integration events into an oncogene or tumor suppressor may cause insertional

**Adenoviral vectors (Ad)** engineered to be devoid of the viral genome were the first to be utilized for CF gene supplementation therapy. These vectors have the advantage of being nonintegrating, with a natural tropism for the lung. In clinical trials using Ad-CFTR, low levels of gene transfer and partial correction of chloride transport in nasal epithelium were observed in some patients [16]. However, issues such as dose-dependent lung inflammation and humoral and cellular immune responses preventing repeat administration remained limiting

**Adeno-associated viral vectors (AAV)** also remain largely episomal inside the nucleus, minimizing the threat of insertional mutagenesis. Over 130 serotypes of AAV have been identified, with each viral capsid demonstrating its own unique transduction profile [18]. Capsids from AAV1, 5, 6, 8, and 9 may be the most efficient for transducing cells of the airway epithelium [19]. In addition, the creation of hybrid AAV capsids, such as AAV6.2, may allow the customization of vectors optimized for transducing the desired target cell. Early phase I trials with AAV2.CFTR showed limited efficacy, due in part to the use of a non-lung-tropic AAV2 serotype, limited packaging space for an optimal promoter (*CFTR* cDNA uses 4.7kb of the vector's ~5kb packaging capacity), as well as AAV capsid-specific immune responses limiting repeat administration [16]. Strategies aimed at minimizing adaptive immunity to AAV vectors or reducing the need for repeat administration continue within the field. Removing CpG motifs from AAV vectors or designing hybrid AAV capsids has been shown to reduce innate and adaptive immune responses following intramuscular delivery [20,21]. Targeting AAV delivery to progenitor cells in mouse lung also shows promise as a means of avoiding

**Lentiviral vectors** based on recombinant human (HIV), simian (SIV), feline (FIV), and equine (EIV) immunodeficiency viruses have also been investigated for gene replacement therapy [16]. Lentiviral vectors are pseudotyped with the envelope proteins from various viruses to increase tissue tropism. The vesicular stomatitis virus G (VSV-G) envelope glycoprotein has most commonly been incorporated, although the F and HN proteins from murine parain‐ fluenza virus type 1, or Sendai virus (SeV), may improve airway transduction. Studies with SeV-pseudotyped lentiviral vectors have accommodated repeat administration to murine airways in pre-clinical studies [23]. Should repeat administration also be feasible in human subjects, the two major remaining limitations to lentiviral use include safety concerns over genomic integration and scale-up of vector production. The concern over vector integration came to the forefront in 2003, when the integration of a retroviral vector used to treat X-linked severe combined immunodeficiency (X-SCID) triggered unexpected activation of a proto-

lung cell turnover and circumventing the need for redelivery [22].

oncogene leading to leukemia in nearly half of the trial's participants [24,25].

mutagenesis leading to cell death or cancer.

362 Cystic Fibrosis in the Light of New Research

factors.

In recent years, transcript supplementation therapy has been introduced as an alternative to gene replacement therapy (Figure 2B). As mRNA transcripts are not capable of integrating into the chromosome, the threat of insertional mutagenesis is completely void. Following uptake via receptor-mediated endocytosis and lysosomal trafficking, mRNA also completely avoids the rate-limiting step of nuclear entry, being translated rapidly and efficiently directly in the cytoplasm [26]. With its naturally short half-life, mRNA transcripts are particularly useful for applications where short bursts of protein expression are desired. However, the addition of chemical modifications mimicking endogenous mRNA modification schemes has increased expression and stability, while decreasing immune responses. One major benefit to the use of chemically modified mRNA is the ability to readminister the vector as necessary.

The use of mRNA itself has long been appealing as an alternative to gene-based delivery vehicles. Unfortunately, for many years researchers were unable to use *in vitro* transcribed mRNAs to upregulate protein expression *in vivo*, as these transcripts were immediately recognized and destroyed by the immune system following injection [27]. Recent work has shown that by completely substituting uridine with pseudouridine during mRNA synthesis, the binding affinity of mRNA to innate immune receptors can be reduced, making systemic *in vivo* application possible [28,29]. More recent work has shown that partial substitution of combinations of various nucleotide modifications, more closely mimicking those observed in endogenous transcripts, can yield mRNA transcripts with further increased stability, specifi‐ cally in murine lung [30,31]. This emphasizes that the design of mRNA may have substantially different effects in specific organs *in vivo* compared with *in vitro* use.

In one recent study, transcript therapy with chemically modified *surfactant protein B (SP-B)* mRNA exhibited success in achieving therapeutic levels of protein expression in a murine model of SP-B deficiency [30]. Repeated intratracheal administration of modified *Foxp3* mRNA to murine lung was also shown to alleviate asthma symptoms in two different models of experimental asthma [31]. Both of these models demonstrate the efficacy of nucleotide modified mRNA in achieving therapeutic levels of protein expression in the lung following repeated, *in vivo* delivery. As a vehicle for delivery, modified mRNA may present a safer alternative to viral and non-viral DNA-based approaches, as immune activation can be efficiently prevented and the possibility of genomic integration is eliminated. Importantly, however, due to the short half-life of mRNA, the benefits of modified mRNA transcripts may be better utilized outside of direct transcript supplementation.
