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Aptamer-enhanced gene therapy delivery

Gene therapy is the future for the treatment of several, often life-threatening conditions. Several gene therapy assets are currently under trial targeting cancers, sensory organs, blood disease, and rare genetic conditions across multiple therapy areas.

Here we examine the potential for aptamer-based solutions to enhance gene therapies and increase the value of these treatments through improved targeting with reduced off-target effects to increase the therapeutic index.

Promising outlook for the gene therapy market

With the global gene therapy market predicted to grow at a CAGR of 68.6% between 2020 and 2026, this sector represents an expanding area of interest for Pharma and Biotech companies alike. Although initial approvals for these therapies have covered relatively small patient groups and rare diseases, the significant pipeline of gene therapeutics is set to expand the impact of these treatments and unleash their true potential.

aptamers for gene therapy

In 2017, the successful approval and ongoing phase 4 trials for Luxturna (Novartis) marked a transition for the field from experimental to commercial. Recognition of the advantages that gene therapy can bring to healthcare systems and patients are spurring further investment in this sector.

Gene therapy advantages and disadvantages

How does gene therapy work?

Gene therapy aims to treat diseases by replacing, inactivating or introducing genes into a patients cells in order to compensate for abnormal gene expression or to make a beneficial protein. Most approved approaches utilize viral vectors to deliver the gene therapy, which can take many forms, such as plasmid DNA, siRNA, and antisense oligonucleotides (ASOs) for gene editing. However, recent developments in clustered regularly interspaced short palindromic repeats (CRISPR) gene-editing technology have increased its potential application in bringing viable gene therapies to the clinic.

To improve transgene delivery gene therapeutics necessitate a vector with good retention time in the body that can cross the cell membrane, preferably in a targeted and specific manner with a triggered release of the transgene payload. A major obstacle that still hampers the development of new gene therapies is the difficulty in directing them to specific target organs.

Delivering gene therapy to a target cell

Often the most critical step in achieving success with a gene therapy is selecting the correct vector to ensure delivery and penetrate the tissue or cell. Delivery of highly charged oligonucleotides, such as siRNA and antisense oligonucleotides, across the cell membrane is challenging, though advancements in this field have shown highly efficient delivery for both viral and non-viral methods.

Aptamer Delivering gene therapy to a target cell

Aptamers to improve viral vector efficiency

Viral vectors are the most commonly used approach to address vector delivery issues. They are known to achieve high transfection efficiencies in the host, but are associated with immunogenicity and cytotoxicity issues. Deletion of the virulence genes for increased safety profiles may affect their ability to infect or integrate with the host chromosome, thus compromising their effectiveness as vectors.

Among current gene therapy studies, adeno-associated viruses (AAVs) are the most commonly used viral vectors due to their high titer, low immunogenicity, and low genomic integration rate. Of the eleven serotypes of AAV they all exhibit different tropism, but with many able to target multiple tissues, resulting in additional requirements for cell-specific targeting of gene therapy. Additionally, AAV efficacy can be negatively affected by the prevalence of neutralizing antibodies in serum, reducing the potential dose of the gene therapy that reaches the target cells.

Aptamers have been explored to enhance gene transduction and expand the tropism of AAV vectors. In a proof-of-concept study, from Professor Weihong Tan’s lab at the University of Florida, conjugation of an AAV2 vector to aptamers targeting the cell membrane protein PTK7 enhanced gene transduction of cancer cells with a fluorescent marker gene. Conjugation of the aptamer to the vector also expanded the tropism of the vector to heparan sulfate proteoglycan-negative cell lines that are typically inaccessible to this vector serotype and increased the efficiency of cellular uptake of the transgene.

Lentiviral insertion of gene therapy transgenes has been extensively explored, particularly in the field of T-cell engineering, due to the ability of lentiviral vectors to stably integrate a transgene into a host genome. Using a lentivirus vector conjugated to an EGFR-specific RNA aptamer, the Center for Biologics Evaluation and Research, showed increased specific cellular targeting of the vectors for aptamer-mediated transduction of the gene therapy.

Increasing the dose with aptamer-targeted non-viral vectors

Non-viral vectors offer biosafety advantages over viral vector approaches. They also benefit from low cost and ease of production, but have been associated with poor delivery efficiency, resulting in low dosing of the gene therapy. Despite the previous evidence of low dosing of gene therapies, the use of non-viral vectors is on the rise, with many researchers engineering improved delivery strategies to support the enhanced safety parameters of non-viral vectors.

Non-viral gene therapy delivery includes both physical and chemical delivery methods. Physical gene therapy delivery methods employ physical force, such as microinjection or electroporation, to facilitate the transport of the gene therapy into the cell. The most commonly used technique is electroporation, though this is limited to the ex vivo editing of cells.

Chemical methods for delivery include the use of nanoparticles to encapsulate the gene therapy and protect them from degradation, target them to specific tissues and potentiate cell uptake and internalization. Many different nanoparticle structures are being investigated, including the use of polymeric- and lipid-based nanoparticles. In many cases targeting the nanoparticle for internalization, particularly into the cell nucleus, can prove challenging. A survey of research over the period 2005-2015 showed that only a median of 0.7% of the systemically administered nanoparticle dose reached solid tumors in murine models. Chemical conjugation of the nanoparticles with different targeting ligands, such as aptamers, has been shown to enhance cellular uptake and specificity.

In one study, led by Peking University’s Professor Zhenjun Yang, scientists used nucleolin-targeting aptamers conjugated to PEGylated liposomes to act as targeting ligands for the delivery of anti-BRAF siRNA, for the treatment of radiotherapy resistant melanoma. This strategy achieved specific cell targeting to the cancer cells and considerable silencing activity, with excellent potential for clinical application for the treatment of melanoma.

Although aptamers as targeting ligands can be conjugated with therapeutic agents such as siRNAs and ASOs directly, an additional advantage of nanoparticles is the ability to deliver larger amounts of therapeutic or diversified therapeutics to target cells per delivery and biorecognition event.

Nucleolin-specific RNA aptamers were also used to target gold nanocage vehicles loaded with doxorubicin hydrochloride and siRNA for a combination of tumor-responsive genetic therapy, chemotherapy and photothermal therapy. Results from a lung cancer mouse model dosed over one month with this combination treatment showed tumor eradication and increased survival of the mice.

Oligonucleotide targeting with aptamers

In addition to targeting vectors carrying a gene therapy payload, aptamers can be conjugated directly to oligonucleotide therapeutics, such as siRNA and ASOs, for targeted delivery. In this capacity, aptamers offer several benefits over alternative targeting molecules, including reduced toxicity profiles, improved manufacture and small size for increased tissue penetration, particularly for hard-to-access sites, like solid tumors.

Aptamers can be readily taken up by cells through a number of mechanisms, most commonly endocytosis and micropinocytosis. Uptake is ultimately determined by the specific interaction with their target, making them very appealing for the delivery of therapeutic oligonucleotides to the cell interior for gene therapy applications.

A study using siRNA conjugated to aptamers specific for the gp120 surface protein of human immunodeficiency virus 1 (HIV-1) capsid administered to mice models over three weeks showed a 105-fold reduction in the concentration of the viral RNA in plasma.

Delivery of therapeutics across the blood-brain barrier (BBB) has long been challenging, resulting in limited treatment options for many brain and central nervous system diseases. Aptamers have shown to be a promising class of therapeutics for the potential treatment of brain disorders, as they are small, non-immunogenic and able to permeate the blood-brain barrier with relative ease, overcoming the need for invasive intrathecal injections for drug delivery. A recent study showed the ability of a transferrin receptor aptamer to facilitate transcytosis across the BBB and deliver an oligonucleotide therapeutic for the inhibition of Tau phosphorylation. Increased brain exposure and ability to disrupt ‘tauopathies’ was observed with this aptamer-based delivery vehicle in mice models

For aptamer-oligonucleotide conjugates, there is the potential to synthesize the aptamer-oligonucleotide therapeutic conjugate as a single contiguous molecule, removing the need for additional conjugation and purification of the final aptamer with the therapeutic cargo. This greatly reduces production costs, increases standardization of the product and improves yields relative to processes requiring post-synthesis conjugation.

Aptamer to Optimer for improved performance and manufacturability

At Aptamer Group, we are experienced in the design and selection of Optimer binders for the targeted delivery of gene therapies, including engineering access to the nucleus for the effective delivery of oligonucleotide and vector-based strategies.

aptamer to optimer

Optimers are smaller oligonucleotide binders that the parent aptamer than the parent aptamer and are optimised for target, performance in the end-use assay and manufacturability. 

To ease the path to the clinic and ensure that your binder displays optimum performance, stability and manufacturability we incorporate additional development steps into each selection project. Rather than use the initial selected aptamer candidate, all our aptamers are further engineered to Optimer binders. This proprietary process involves analysis of the selected aptamer candidate to identify the smallest fragment that possesses the desired binding characteristics. Production of Optimer binders results in smaller molecules, increased stability, can improve target affinity, and results in more cost-effective manufacture of the final Optimer.

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