Gene-Targeted Therapies: Overview and implications

Abstract

Gene-targeted therapies (GTTs) are therapeutic platforms that are in principle applicable to large numbers of monogenic diseases. The rapid development and implementation of GTTs has profound implications for rare monogenic disease therapy development. This article provides a brief summary of the primary types of GTTs and a brief overview of the current state of the science. It also serves as a primer for the articles in this special issue.

1. Introduction

The goal of this contribution to the special issue is to provide a general overview of gene-targeted therapies (GTTs) and to set the stage for the accompanying papers. Common types of GTTs are described and a comparison is made between traditional therapeutic approaches and those of GTTs. This information is not intended to be an in-depth review of the state-of-the-art of GTTs. However, references to recent review articles are provided. Background information on some of the terms used herein can be found at https://www.genome.gov/genetics-glossary.

2. Traditional drug development versus gene-targeted therapies

Prior to the era of GTTs, the development of therapeutics for monogenic disease started with and required an understanding of the biochemical abnormality of the disease. This by itself was a difficult and time-consuming process. Only after the biochemical abnormality was identified, could the quest for a therapeutic begin in earnest. Typically, this would involve screening of large numbers of small molecules for therapeutic efficacy followed by optimization of lead compounds for those with favorable pharmacokinetic, pharmacodynamic, and toxicological profiles. Recent developments in high throughput screening and computational in silico drug design have certainly made the process more efficient, but it remains largely empirical and therefore time-consuming and expensive. Notably, the fact that the disease is monogenic in origin was of no relevance to therapeutic development because the genetic material (DNA and RNA) was not accessible as a therapeutic target.

Nonetheless, effective treatments and therapeutic approaches have been developed for monogenic diseases. An early example is the use of a phenylalanine-deficient diet for patients with phenylketonuria, which is effective in preventing the development of neurological abnormalities from this metabolic disease (Gree, 2021), yet challenging for patients to maintain adherence. Indeed, the effectiveness of this approach provides a compelling rationale for the inclusion of PKU in virtually all newborn screening panels. In the case of enzyme deficiencies, enzyme replacement therapy is another therapeutic strategy which has been successful (Brady, 1983; Dornelles et al., 2021). The main point is that the development of these drugs was dependent upon understanding the biochemical abnormalities in individual diseases, and for the most part represent the “one disease at a time” approach.

In contrast, GTTs are fundamentally different, in that they target the genetic material itself (either DNA or RNA). As such, they are inherently therapeutic platforms which can at least in principle be readily adapted to different diseases. The key characteristic of GTTs for the present discussion is that they can be designed based solely upon knowledge of the causative genetic variation underlying the disease, as well as knowledge of the cellular target. No information about the biochemical or cellular abnormalities underlying the disease is required to design a GTT, although such knowledge is important to ultimately test the efficacy of candidate GTTs in vitro and ultimately in vivo. Table 1 shows a comparison between GTTs and traditional drug development.

Table 1.

Traditional monogenic therapy vs gene targeted therapy

Traditional monogenic disease therapy development	Gene-targeted therapies
requires knowledge of biochemical defect	knowledge of biochemical defect not required
knowledge of disease-causing genetic variant is not required	Design is based upon knowledge of the sequence of the disease-causing genetic variant
PK/PD and biodistribution must be optimized for each new molecule	standardized PK/PD and biodistribution for each type of GTT
for small molecule drugs, cost of production may be quite low	cost of production is high, especially for gene therapies based on viral vectors
development is typically "one disease at a time "	therapeutic platforms applicable to multiple monogenic diseases
one drug for one disease is a standardized, predictable regulatory pathway	regulatory pathways evolving rapidly, especially for therapeutic platforms

The transition to gene targeted therapies was made possible by advances in DNA sequencing, which allowed for identification of disease-causing mutations, as well as the technological advances in gene targeted therapies, as outlined below.

3. Types of gene-targeted therapies

A. Gene Therapy

As used in the accompanying articles, the term gene therapy, or vector-mediated gene addition, relates to the delivery of a therapeutic gene construct into the cell types most severely affected in the disease.

There are two main types of gene therapy: in vivo and ex vivo (Figure 1). In vivo gene therapy delivers new or corrected genes using a viral vector directly into the body either systemically or locally. In vivo gene therapy can be used to treat diseases impacting the liver, muscle, and central nervous system, including the eye. At the present time, the most commonly used vector for in vivo gene therapy is based on recombinant adeno-associated virus, or rAAV (Kuzmin et al., 2021). Wild-type AAV is a small, single stranded DNA virus which by itself is generally considered to be non-pathogenic. To construct rAAV gene therapies, the viral genes are removed, and replaced with the therapeutic gene construct of interest, typically including the protein coding sequence and regulatory elements. There are now three FDA-approved products using AAV for gene therapy to treat monogenic diseases: Luxturna for RPE65-mutation associated retinal dystrophy, Zolgensma for spinal muscular atrophy (Au et al., 2021) and most recently Hemgenix (etranacogene dezaparvovec) for hemophilia b (U.S. Food & Drug Administration 2022b).

Figure 1.

In vivo and ex vivo gene therapy.

In ex vivo gene therapy, a viral vector (most commonly based on lentiviruses) is used to deliver a working copy of the defective gene into the genomic DNA of the patient’s hematopoietic stem cells (HSCs) obtained from the patient’s blood sample. Following a course of chemotherapy, which is used to deplete the endogenous pool of HSCs, the gene-corrected stem cells are then infused back into the patient (Consiglieri et al., 2022). When successful, these gene-corrected stem cells proliferate and repopulate the stem cell niche. To the extent that this happens, the treatment can have a therapeutic benefit.

Ex vivo gene therapy is most commonly used for the treatment of hematological and immune disorders (Ferrari et al., 2021), but can also be used to treat neurologic manifestations of lysosomal storage disorders. This is possible because microglial cells in the brain actually derive not from neural stem cells but from HSCs. The rationale for this treatment, therefore, is that after the chemotherapy, infusion of gene-corrected HSCs can result in cells crossing the blood-brain barrier, where they then can differentiate into microglial cells (Biffi et al., 2011). For some diseases, these gene-corrected microglial cells can then secrete enzymes which are taken up by surrounding brain cells to have a therapeutic benefit. An ex vivo gene therapy for cerebral adrenoleukodystrophy (Elivaldogene autotemcel) was approved by the European Medicines Agency in 2021, although the authorization was withdrawn by the sponsor in 2022 (European Medicines Agency, 2022).

In the US, the FDA has very recently approved Zynteglo (betibeglogene autotemcel), for the treatment of adult and pediatric patients with beta-thalassemia (U.S. Food & Drug Administration 2022). Other examples of FDA-approved ex vivo gene therapy products cancers include Kymriah (U.S. Food & Drug Administration 2017b), a genetically modified autologous T-cell immunotherapy. This treatment was approved in 2017 for certain pediatric and young adults with a form of acute lymphoblastic leukemia (ALL). Another example is a CAR T-cell therapy, brexucabtagene autoleucel (Tecartus) (U.S. Food & Drug Administration 2021a) for patients with mantle cell lymphoma, based on the ZUMA-2 clinical trial (Wang et al., 2020).

B. Gene editing

Gene editing involves the delivery of enzymes that cut the target DNA or otherwise modify the target DNA to correct pathogenic mutations (Figure 2). The earliest gene editors were nucleases, which are fused to sequence-specific DNA binding modules for sequence-specific targeting (Kim & Kim, 2014). In the CRISPR-Cas9 system (Knott & Doudna, 2018), the Cas9 nuclease is targeted within the genome using guide RNAs. The original Cas9 and its derivatives created double strand breaks in the DNA but led to off-target breaks. More recently developed adaptations of this system remove the double strand nuclease and instead fused other DNA repair enzymes to the nuclease-deficient Cas9 protein. These “second-generation” Cas9 proteins include the base editors, which can correct single base mutations in DNA. The original base editor (Komor et al., 2016) was designed to correct C-> T mutations in DNA, which represent approximately 45% of all single base mutations that cause human disease. The subsequent discovery of additional base editors (Gaudelli et al., 2017) further increased the ability of Cas9 derivatives to correct single base mutations. Variants of these base editors have also been discovered that act on RNA rather than DNA (Porto et al., 2020).

Figure 2.

Gene Editing. From NIH Gene Editing – Digital Media Kit (National Institutes of Health)

While the base editors represented a major step forward in genome editing, this approach was limited to the correction of transition mutations. The subsequent discovery of the prime editor represents a major step forward, because the incorporation of a reverse transcriptase step expands the types of mutations that can be corrected to nearly 90% of mutations that cause human disease (Anzalone et al., 2019). More recent improvements on the prime editor have increased the versatility of the system even further (Anzalone et al., 2022; Hosur et al., 2022).

It is important to point out that all of the discussions related to gene editing in this special issue focus exclusively on somatic gene editing, meaning therapies that edit the somatic cells (cells other than sperm or egg cells) in an individual. Changes in the genome of somatic cells cannot be passed on to future generations. In contrast, germline gene editing, i.e., editing of an embryo or sperm or egg cells (germ cells), results in changes in the genome that could be transmitted to future generations. There is an international moratorium against the clinical application of germline editing (Lander et al., 2019). All discussions of gene editing in this special issue therefore refer exclusively to somatic gene editing.

C. Oligonucleotide therapies

Oligonucleotides are short pieces of DNA or RNA that bind to specific molecules of RNA and can block the cell’s ability to make protein or function. They are able to modulate gene expression via a range of processes including RNAi, target degradation by RNase H-mediated cleavage, splicing modulation, non-coding RNA inhibition, gene activation and programmed gene editing (Roberts et al., 2020).

Oligonucleotides function in either a sequence-dependent (antisense oligonucleotides [ASOs] and immune stimulatory oligonucleotides [ISOs]) or tertiary structure-dependent manner (e.g., aptamers) (Scharner & Aznarez, 2021). Oligonucleotide therapies include small interfering (siRNA) and antisense oligonucleotides (ASOs). SiRNA therapeutics act by triggering the targeted degradation of RNA molecules by activating the endogenous RNA interference pathway (Gangopadhyay & Gore, 2022). ASOs act by hybridizing to target RNA molecules to either prevent translation of mRNA into protein, or by blocking disease-causing RNA splicing reactions (Spitali & Aartsma-Rus, 2012) (see figure 3).

Figure 3.

Antisense Oligonucleotide Top panel: An intronic mutation results in a cryptic splice site, resulting in the retention of intronic sequence in the mRNA. An ASO blocks this site, resulting in correct splicing.

Bottom: A mutation within exon B results in only a portion of exon B being included in the mRNA. The ASO blocks this site, resulting in restoration of correct splicing. These are two examples of how ASOs can be used therapeutically. Exons are drawn as colored rectangles, introns as orange lines. ASOs and mRNA diagrams not drawn to scale. Adapted from (Hammond & Wood, 2011).

An early oligonucleotide therapy that was approved by the FDA in 2013, was Kynamro (mipomersen sodium) (U.S. Food & Drug Administration 2013) for the treatment of Homozygous Familial Hypercholesterolemia. More recently, Viltepso (Viltolarsen) (U.S. Food & Drug Administration, 2020b), in 2020 and AMONDYS 45 (Casimersen) (U.S. Food & Drug Administration, 2021b) in 2021 were approved by the FDA to treat Duchenne muscular dystrophy. These are both examples of ASOs.

Examples of approved siRNA include Givlaari (Givosiran) (U.S. Food & Drug Administration, 2019) approved in 2019, for use in Acute Hepatic Porphyria and Oxlumo (Lumasiran) (U.S. Food & Drug Administration, 2020a) in 2020, for the treatment of Primary hyperoxaluria type 1.

Overview:

One notable aspect of developing treatments for monogenetic disease is that the root cause, i.e. mutations in the sequence of the genome, are known. It follows that GTTs which correct these mutations can, at least in principle, treat, cure, or even prevent the onset of disease. In practice, the clinical impact of GTTs will depend upon multiple factors, including how many cells in clinically relevant tissues are corrected, as well as the stage of the disease at the time the treatment is administered. Importantly, GTTs cannot replace cells that have been lost in degenerative diseases, nor can they reverse developmental abnormalities. At the present time, GTTs are at a very early stage in clinical development, and much remains to be learned about safety and efficacy. However, to maximize the future clinical benefit of GTTs, the early and equitable diagnosis of all patients who might benefit from them is essential.

4. Summary

In contrast to traditional drug development, GTTs are therapeutic platforms that are in principle applicable to large numbers of monogenic diseases. The rapid development and implementation of GTTs has profound implications for the way we think about developing treatments for monogenic diseases, which in turn has major practical implications for how we identify all patients with such diseases who might benefit from GTTs, in an equitable manner, as early as possible. These issues and others will be addressed in other contributions to this special issue.