Alternative gene therapies could be effective in treating some rare diseases

By Blake Middleton, The Science Advisory Board contributing writer

August 11, 2021 -- Bacteria-mediated genetic transfer has emerged as an alternative gene therapy (AGT) for the treatment of some rare diseases, such as phenylketonuria (PKU). This type of therapy is advantageous because it is easily regulated through established protein expression systems. One company, Synlogic, has developed an AGT candidate that uses a bacterial vector to treat PKU.

The precise classification of therapeutics is not always straightforward. While some approaches fall squarely within the definitional boundaries of gene therapy, others around the periphery are less clear-cut. A class of bacterial therapeutics that fall into this gray area has been called alternative gene therapy (AGT).

Unlike more conventional bacterial-mediated genetic transfer (a technique called bactofection), AGT involves no actual gene transfer to host cells. Instead, the task of manufacturing therapeutic proteins is offloaded entirely to the engineered bacterium, which can either reside outside the target cells or be engulfed by them.

The main advantage of AGT compared to conventional approaches is its ease of regulation. Protein expression can be regulated with inducible bacterial promoters using small molecules, and treatment can be terminated immediately by the application of antibiotics. Auxotrophic modifications can also be made to limit the organism's ability to grow without special nutrients.

AGT is also known as bacterial protein delivery, which is perhaps a more precise and informative term. Synlogic specializes in this approach, calling it "synthetic biotic" therapy. The company's recent work in developing a therapy for PKU illustrates the platform's key features.

A history of PKU therapy

PKU is an inherited metabolic disorder caused by mutations to the PAH gene, resulting in abnormally low levels of the enzyme phenylalanine hydroxylase (PAH). PAH catalyzes the hydroxylation of phenylalanine to form tyrosine, and lack of the enzyme leads to the buildup of toxic levels of the amino acid phenylalanine. It is an autosomal recessive disorder (meaning both inherited copies of the gene must be defective), and it affects about 1 in 12,000 people.

Untreated, PKU can lead to a variety of neurological problems, including the following:

  • Intellectual disability
  • Behavioral problems
  • Seizures and tremors
  • Cognitive problems
  • Mental disorders

Treatment for PKU typically involves carefully following a restrictive diet to avoid foods with high levels of phenylalanine. This regimen can be difficult to comply with, as phenylalanine is an essential amino acid commonly found in milk, eggs, soybeans, and most meats (including fish and shellfish). The artificial sweetener aspartame is a dipeptide of the amino acids aspartic acid and phenylalanine.

Early detection of PKU is essential, as are regular blood tests, to determine optimum dietary requirements. Because phenylalanine is required for production of most proteins, minimum phenylalanine levels must be maintained. Infants are screened at birth in most developed countries so that treatment may begin immediately.

More recently, enzyme replacement therapy (ERT) has been developed to treat PKU. One of the drawbacks of the PAH enzyme is its poor stability, so research has instead focused on more stable enzymes that have the ability to break down phenylalanine. Phenylalanine ammonia lyase (PAL), found in many plants, fungi, and bacteria, is one such alternative. PAL catalyzes the transformation of phenylalanine to ammonia and trans-cinnamic acid (TCA), which can then be converted to hippuric acid and excreted in urine.

In 2018, the U.S. Food and Drug Administration approved BioMarin's enzyme substitution therapy pegvaliase (Palynziq) to treat adults with PKU. Pegvaliase contains PAL derived from Anabaena variabilis, a cyanobacterium. Because the bacteria-derived PAL is strongly immunogenic, the PAL enzyme in pegvaliase is bound with polyethylene glycol to block immunogenic epitopes in a process called PEGylation. Despite these modifications, the drug can still cause severe allergic reactions, complicating its administration.

An oral version of PAL could solve many of the challenges presented by subcutaneous administration and would have much broader therapeutic potential. Dietary phenylalanine is present in the human gastrointestinal (GI) tract, so an oral formulation of PAL could reduce phenylalanine concentrations before the amino acid has even been absorbed into the body.

Evidence also suggests that the body's systemic phenylalanine is continually recirculated through the gut, further raising an oral formulation's therapeutic potential. Unfortunately, despite PAL's enhanced stability compared to PAH, it cannot survive long enough in the GI tract to have therapeutic effect, and efforts to stabilize the enzyme for oral administration have not been successful.

A new approach to treating PKU

Synlogic's approach uses a bacterial vector to manufacture phenylalanine-metabolizing enzymes within the GI tract. SYNB1618 is a genetically engineered strain of a probiotic bacterium (Escherichia coli strain Nissle 1917), which has been modified to include multiple copies of two gene-encoding enzymes that degrade phenylalanine:

  1. PAL, an enzyme derived from the bacterium Photorhabdus luminescens, which converts phenylalanine to ammonia and TCA
  2. L-amino acid deaminase, an enzyme derived from the bacterium Proteus mirabilis, which converts phenylalanine to phenylpyruvate in the presence of oxygen

The strain's ability to take up phenylalanine is also enhanced with additional copies of one of its endogenous genes, encoding a high-affinity transporter of phenylalanine called phenylalanine-specific permease. Finally, a gene essential for growth has been deleted from SYNB1618, rendering it unable to grow without special supplementation. (The dapA gene encodes an enzyme called 4-hydroxy-tetrahydrodipicolinate synthase, which is necessary to synthesize a chemical called diaminopimelate.)

Phase I/IIa clinical trial results for Synlogic's SYNB1618 therapeutic were recently published in Nature Metabolism, and the data showed that the drug was both safe and well tolerated. The organism was also unable to colonize the GI tract, and SYNB1618 completely cleared within four days of dosing. Breakdown products of phenylalanine were detected in higher concentrations in patients' urine and plasma, but the overall therapeutic effect on patients' phenylalanine levels is unclear.

Synlogic is working on developing other synthetic biotic therapies, including SYNB1934, a further refinement of their PKU treatment; SYNB8802 to treat enteric hyperoxaluria, an acquired metabolic disorder; and SYNB1891, an immunotherapeutic targeting solid tumors by activating the stimulator of interferon genes (STING) pathway.

Blake Middleton is the editor of Cell and Gene Therapy Business Outlook, part of Science and Medicine Group.

Disclosure: Cell and Gene Therapy Business Outlook is a sister publication of ScienceBoard.net.

Do you have a unique perspective on your research related to gene therapy? Contact the editor today to learn more.

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