Proposed Implementation
SCUT_China Introduction
Briefly, we constructed two phage mutants in our project. The first mutant T_S1 was obtained by inserting relE gene within the ORF73 of S1, a hypothetical protein, while RelE inhibits protein synthesis by cleaving mRNA codons on the ribosomal A site in a sequence specific way with preference for the stop codon UAG(Pedersen et al., 2003) and has great inhibitory effect on bacterial growth in vitro and in vivo(Pedersen, Christensen, & Gerdes, 2002). After editing by CRISPR-Cas9, all the plaques testes contained recombinant phages with desired mutation. For further biosafety consideration, we constructed a lysis-deficient phage (LyDT_S1) expressing the antibacterial toxin RelE following the DBTL (design→build→test→learn/redesign) engineering cycle. The mutant was obtained by replacing the gene of holing, a lysis-promoting phage toxin, with the relE gene. In lysis kinetics for characterization of bacteriostasis effect, high MOI (multiplicity of infection) group of mutants indicates that bacteria do not develop resistance to phages. It might help solve one of the main questions regarding phage therapy，the possible rapid emergence of phage - resistant bacterial variants, which could impede favourable treatment outcomes (Oechslin, 2018; Rohde et al., 2018).
After editing, the mutant phage LyDT_S1 can still inhibit bacterial growth through the expression of RelE but cannot induce cell lysis because the holin gene is eliminated. This is expected to reduce the amount of endotoxins released from the pathogen bacteria, with a corresponding reduction of systemic cytokine response and inflammation during bacterial infection. The dilution & purification and endotoxin release testing of LyDT_S1 are still under way.
In proof of concept, we only writed the research plan due to the impact of the outbreak on the season: (1) Pharmacokinetics and Pharmacodynamics of T_S1 & LyDT_S1. (2) Validation of the Effects of T_S1 and LyDT_S1 on Endotoxin and Inflammatory Mediators Release in a Murine Bacteremia Model
In conclusion, we hope that our engineered phages can actually be applied to phage therapy in the future and all of us struggle with it from time to time. In our proposed implementation, we integrated and analyzed existing phage products, and compared them with our expected engineered phage products. Then, we collected relevant laws and regulations on phage drugs to stimulate the development of products. Last but not the least, we discussed Obstacles & Challenges to phage treatment and put forward corresponding suggestions.
Hopefully our work will be of value to others who take similar routes later on. Ⅰ. Summary analysis of existing products
In the proposed implementation, we hope to extend our research results to the production of phage therapy drugs. We first conducted product investigation on domestic and foreign companies engaged in phage therapy drug development to understand the main forms and production processes of existing phage drugs, so as to gain valuable experience in phage drug development Table 1).
After the survey was completed, we found the products most similar to our expected products for comparison （Table 2) and proposed our implementation draft.
We collected relevant laws and regulations on phage drugs (Table 5), hoping to standardize our production pipeline, make our expected products conform to relevant laws and regulations, ensure product safety, and provide future users with better experience.
Next, we collected relevant data on phage therapy NGOs (Table 3) and related research centers (Table 4), hoping that the work of these organizations and centers will inspire us to open up diversified implementation paths.
i.Compassionate phage therapy
There is a current unmet medical need for the treatment of antibiotic-resistant infections, and in the absence of approved alternatives, some clinicians are turning to empirical ones, such as phage therapy, for compassionate treatment. However, compassionate phage therapy (cPT) remains limited to a small number of experimental treatment centers or associated with individual physicians and researchers, which exposes these problems: subsequent steps are difficult to scale, and treatment paradigms stand highly variable between cases, or are frequently not reported.
Compassionate treatment denotes the use of unapproved medicines outside of clinical trials for the treatment of patients for which approved therapeutic options have been exhausted. The principle of compassionate use is codified in the “Helsinki Declaration of Ethical Principles for Medical Research Involving Human Subjects”, which is an international agreement on facets of clinical research, such as patient consent and placebo control (General Assembly of the World Medical, 2014). Article 37 specifically asserts a physician’s authority to act in the best interest of their patient by using experimental treatments in the absence of approved options, although the support of using unproven treatments was not stipulated by the Declaration until its amendment in 2000 (v2000, Article 32). The intention of compassionate use is to increase therapeutic options for patients and highlight the inability of current pathways to respond punctually to medical needs.
cPT has been approved under emergency investigational new drug (eIND) and expanded access schemes by the FDA, a temporary use authorization (ATU) by the French National Agency for Medicines and Health Products Safety (ANSM) in France, by special access schemes by the TGA in Australia, and by national regulation in Poland. And for physically- and financially-able patients without local support, they can travel to countries countries where it is an approved medicine, such as the Eliava Institute in Tbilisi, Georgia(Fadlallah, Chelala, & Legeais, 2015; Patey et al., 2019; Zhvania, Hoyle, Nadareishvili, Nizharadze, & Kutateladze, 2017). The exact process for organizing cPT remains highly variable and delay the initiation of treatment and influence therapeutic outcomes (Figure 1).
Figure 1. cPT workflow and an example of the Queen Astrid military hospital in Brussels (Belgium)(Djebara et al., 2019)
We hope that peers and related patient groups will work together to promote the popularization of phage compassion therapy for the benefit of more people who attempts to use conventional treatment have been exhausted, and there are no active clinical trials suitable for enrolment.
ii.Trial design standardization report.
The instances of cPT are presented at conference, published as press release and publication, but lack a more standardized reporting guidelines to make comparisons between treatments concerning treatment outcomes, concomitant antibiotic use, phage-resistant variant microbiological assessment, the treatment of a myriad of different indications for both chronic and acute conditions and etc. Particularly, even definitions of clinical “success” or “failure” may vary, therefore cautioning against the over-interpretation of some cPT results.
We have collected some treatment cases for future development and application of engineered phages, taking P. aeruginosa (Aslam et al., 2018; Chan et al., 2019; Fadlallah et al., 2015; Jennes et al., 2017; Khawaldeh et al., 2011; Patey et al., 2019; Ujmajuridze et al., 2018; Zhvania et al., 2017), S. aureus (Fadlallah et al., 2015; Randolph Fish, Kutter, Bryan, Wheat, & Kuhl, 2018; R. Fish et al., 2016; Jikia et al., 2005; Leszczynski et al., 2006; Zhvania et al., 2017), A. baumanii (LaVergne et al., 2018; Schooley et al., 2018)as examples. It is just a preliminary clinical data collection and literature reference. In the future, we hope to unit other forces will work together to build a clinical database of phage therapy, such as Shanghai Institute of Phage Therapy and Literature Information Center of Chinese Academy of Sciences, to promote a progress of human medical civilization in the post-antibiotic era.
Furthermore, the standard operation procedure for phage therapy in clinical practice should be explored, including enrollment of patients for phage therapy; establishment of phage libraries; pathogenic bacterial isolation and identification; screening for effective phages against pathogenic bacteria; phage formulation preparation; phage preparation administration strategy and route; monitoring the efficacy of phage therapy; and detection of the emergence of phage-resistant strains(Cui, Guo, Feng, & Li, 2019), for the success of phage therapy in future.
iii.Phage repositories & Protection of intellectual property
The requests of cPT might be dismissed because no suitable phages were available to treat the causative bacterial agents. The public phage repositories are required to establish across international borders with hundreds of potent and characterized phages to provide a timely and effective phage for patients. Factor that lead to conflict is , the phages sourced from academic labs and phage banks can offer the benefit of characterization, including genome sequence to prove no virulence factors or antibiotic genes, host range analysis and in vitro/ in vivo studies to support their use for phage therapy, which are liable to intellectual property(IP) constraints with different degrees and require a material transfer agreement(MTA) that limits and delineates the use of the phages supplied only for emergency treatment.
iv.Phage Resistant
One of the main questions regarding phage therapy is the possible rapid emergence of phage-resistant bacterial variants, which could impede favourable treatment outcomes. Experimental data has shown that phage-resistant variants occurred in up to 80% of studies targeting the intestinal milieu and 50% of studies using sepsis models. Phage-resistant variants have also been observed in human studies, as described in three out of four clinical trials that recorded the emergence of phage resistance(Oechslin, 2018; Torres-Barcelo, 2018). Our project may help with this challenge, but further in vivo test is needed.
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