Gene Therapy and Emerging Molecular Therapies

Principles of Gene Transfer

The phases of nucleic acid delivery, gene expression, action of the newly produced protein, and consideration of adverse effects of gene delivery vectors and gene products are analogous to issues in conventional drug therapy. Development of molecular therapy using nucleic acid begins with the identification and cloning of a gene. The choice of DNA sequence to be transferred is typically a cDNA sequence containing its entire protein coding sequence but may include introns, nuclear localization, or protein secretion signals. In addition, DNA must also contain transcriptional regulatory sequences, a transcription start site, and an RNA polyadenylation sequence to transcribe and stabilize mRNA. These DNA sequences are linked into a single unit that is inserted (subcloned) into a circularized piece of DNA called a plasmid. Plasmids contain genetic sequences that allow replication within bacteria so that large quantities of DNA can be produced and purified. The plasmid containing the therapeutic DNA can be delivered to target cells using one of the many vehicles (“vectors”) for gene transfer. Intracellular transfer of most full-length human genes and associated regulatory elements (that may be more than 100 kilobases; kb) is theoretically optimal but limited by current technology.

Basic concepts of gene transfer and gene expression are similar, regardless of the vehicle used to carry genetic material to the target cell (Fig. 5-1). After administration of the DNA-vector, the vehicle carrying DNA enters the cell by passing through the cell membrane or by active uptake via a specific receptor. The DNA is taken into the nucleus, where it can be processed. The host cell supplies enzymes necessary for transcription of the DNA into messenger RNA (mRNA) and translation of the mRNA into protein within the cytoplasm. The protein functions intracellularly or extracellularly to replace a hereditary deficient or defective protein, or to provide a therapeutic function. The protein may: (1) function intracellularly, such as adenosine deaminase (ADA) used to correct the mutation in lymphocytes responsible for one form of the severe combined immunodeficiency disease (SCID) syndrome; (2) replace a cell membrane protein, such as the cystic fibrosis (CF) transmembrane conductance regulator chloride channel (CFTR) mutant in CF; or (3) introduce a secreted protein, such as factor VIII, which is deficient in hemophilia. Successful gene transfer (also called transfection) and expression are evaluated by measuring RNA, protein, and, importantly, function in the target cell. In contrast to traditional pharmacological approaches, the goal of this therapy is alteration of the genotype of the cell, rather than alteration of the functional phenotype only.

FIGURE 5–1 Generalized cellular schema of gene transfer. A vector carrying an exogenous gene coding for a secreted protein is taken up by a target cell and transferred to the nucleus, where the DNA is transcribed to mRNA. The mRNA travels to the endoplasmic reticulum, where it is translated to protein. The therapeutic protein is then secreted into the circulation.

Nucleic acids can also be delivered into the cell to interrupt specific mRNA translation and subsequent protein production. The two general classes of nucleic acids, using independent mechanisms to silence genes through binding and triggering destruction of targeted mRNA, are antisense and RNA interference (RNAi) (Fig. 5-2). Antisense oligonucleotides are 12 to 28 nucleotide, single-strand sequences that are chemically modified to enhance their t_1/2. Binding of these oligonucleotides to a complementary mRNA sequence results in cleavage of the targeted mRNA by endogenous RNaseH, an endoribonuclease that specifically recognizes RNA-DNA heteroduplexes. The cleaved mRNA and oligonucleotide are degraded, and protein translation is reduced. However, a high abundance of oligonucleotides is required for efficient antisense silencing. Larger oligonucleotides can also be engineered as a ribozyme to bind and directly cleave mRNA, and then repeat this process without self degradation. More than 50 different antisense oligonucleotides and ribozymes are in clinical trials, primarily directed toward oncogenic genes and infectious virus RNAs.

FIGURE 5–2 Mechanisms of RNA interruption. Left, Antisense mechanism. Antisense oligonucleotide enters cell and binds complementary mRNA sequence in the nucleus and cytoplasm. The oligonucleotide-RNA duplex is recognized and degraded by RNase H. Right, RNA interference mechanism. Double-strand RNA is chopped by the enzyme Dicer into 21 to 25 nucleotide siRNAs. siRNA is incorporated into a nuclease complex called the RNA-induced silencing complex (RISC) that unwinds the double-strand RNA. Complementation of the siRNA antisense strand sequence and the target mRNA result in nuclease enzyme binding and cleavage.

A recently developed mRNA silencing system is RNAi that takes advantage of endogenous RNA regulatory pathways (see Fig. 5-2). Large, double-stranded RNA sequences designed to target endogenous mRNA enter the cell and are cut into short interfering RNA (siRNA) by the dicer enzyme. Alternatively, synthetic siRNA may be delivered to the cell directly. In either case, the double-strand siRNA molecules are bound by a group of proteins called the RNA-induced silencing complex (RISC). The RISC proteins activate unwinding the siRNA to single-strand RNA that binds a specific mRNA molecule. The RISC cuts the mRNA in the region paired with the antisense siRNA sequence, and the cleaved mRNA is degraded to prevent protein translation. Clinical trials are in development using RNAi.

Prerequisites for Human Gene Therapy

Application of gene transfer principles to gene therapy requires several critical considerations (Box 5-1). First, a candidate disease for gene therapy must be selected. Typically, this is a disease not successfully treated by currently available therapies. Second, the genetic basis of the disease must be determined by identifying the gene that encodes for a mutant protein or by locating the mutant gene using classical genetic studies. Third, the pathophysiology of the disease must be known so that the cellular site of normal and abnormal gene expression can be ascertained to target the therapy. A corollary is that the magnitude and duration of exogenous gene expression likely to ameliorate the disease should be estimated. Fourth, tools for detection of gene expression must be in hand, including methods for detection of RNA, protein, and protein function. Fifth, preclinical in vitro and in vivo systems for testing efficacy of gene transfer must be developed. This usually mandates that an animal model of disease be available. With few exceptions, these steps are developed in the laboratory before a strategy for clinical gene therapy is considered. Finally, pharmaceutical-grade nucleic acid or virus vector must be produced free of biological and chemical contaminants.

DNA and RNA Targeting and Delivery

How therapeutic DNA is transferred to a specific target cell depends on the biology of the target cell and the vector. Targeting mechanisms and different features of these approaches can be used in combination. The procedure of gene transfer can take place in cells removed from the body (ex vivo), then reintroduced into the patient, or by direct delivery of DNA or RNA to the patient (in vivo).