Directed evolution for aging

UPDATE 06/30/2017: It’s over. I’ve shut this project down, dissolved the company, returned all money back to investors, and removed the names of those who no longer want to be associated with this. I’m keeping much of the rest of this proposal online. I hope that someone better connected than I can find use in this. I’ve written up a full post-mortem of the project, which you can read here: 100 Lessons Learned from Shutting down my Anti-Aging company

1. Introduction

1.1. Aging and Age-related Diseases

Many industrialized nations are facing aging populations coupled with declining birth rates, posing a litany of economic costs and health problems (Kuller et al., 2016). With aging comes exponentially increasing risk of countless diseases such as cancer, diabetes, heart disease, neurodegenerative conditions, osteoarthritis, and gastrointestinal disease. Many of the advances in medical technologies have done little to address this problem. Even over the past 10 years, the age-adjusted death rate due to cancer has barely changed while the total death rate due to cancer has risen, and both the age-adjusted rate and the number of diabetes deaths have increased. Despite many medical advances, the needle has barely moved on the death rate due to Alzheimer’s over the past decade (Johnson, et al. 2014). In response to all of this, more researchers and institutions are investigating the causes of the aging process in the hopes that this understanding could lead to treatments for conditions like heart disease, cancer, Alzheimer’s, diabetes, and many others.

1.2. Epigenetics, Sirtuins, and NAD+

One of the contributors to biological aging is the accumulation of epigenetic errors. The epigenetic model of aging can be explained as follows: The cells in multicellular organisms all have the same DNA (save for immune cells), but some genes will be expressed in some places and not in others because of epigenetic modifiers. Sirtuins maintain these expression patterns, but over time the sirtuins don’t carry out their function as well and genes are inappropriately turned on or off (Revollo et al., 2004). One of the reasons for this is the decrease in NAD+, the cofactor that fuels the sirtuins’ activity. As organisms age, the ratio of charged NAD+ to the uncharged and unused NAD(H) shifts in the direction of the latter, effectively depleting the fuel for the sirtuin proteins (Schultz et al., 2016). This breakdown of sirtuin function is associated with many different health problems such as neurodegeneration, hearing loss, cardiovascular disease, β cell dysfunction, hepatic steatosis, increased adiposity, insulin resistance, dysregulated hepatic glucose, homeostasis, shortened lifespan, and many cancers (Hall et al., 2013; Imai and Guarente, 2014). A popular hypothesis, that is supported by increasing amounts of experimental evidence, is that if cells were resupplied with NAD+ molecules that fuel and activate sirtuins, more of this damage could be repaired. Since NAD+ has difficulty getting into cells due to its charged state, many have tried using molecules that are converted into NAD+ when they’re inside the cell. (Billington et al., 2008; Bonkowski and Sinclair, 2016)

1.3. Supplementation of NAD+ Precursors and Reversal of Aging

In 2013, the Sinclair Lab at Harvard Medical School gave the NAD+ precursor, nicotinamide mononucleotide (NMN), to 22-month old mice (roughly the equivalent of a 60-year old human) over the course of a week. When compared with old mice that had not receive the treatment, the treated mice had muscle mitochondria resembling those of younger 6 month-old mice (roughly the equivalent of a 20 year-old human) (Gomes et al., 2013).

1.4. Effects of Increased NAD+ Levels on Specific Diseases

Additional research has demonstrated that increased NMN levels show promising preliminary results for specific diseases. In studies done at Washington University St. Louis, NMN has been shown to reduce symptoms of both age-related and diet-related diabetes in mice (Yoshino et al., 2011). In cell culture, neurons with increased NMN levels show greater resistance to the kind of axon degeneration common in diseases like Parkinson’s and Alzheimer’s (Araki et al., 2004; Long et al., 2015). Mice with NMN in their drinking water showed reduced symptoms of vascular dysfunction(Picciotto et al., 2016). Data from these studies demonstrate that supplementation with NMN to boost cellular levels of NAD+ may be a novel therapeutic approach for the treatment of a wide range of diseases..

1.5. Problems with Current Production and Delivery Methods

While the role of NAD+ precursors in reversing the effects of aging and other diseases is still under investigation, the high cost of NMN greatly hinders future research. Compared to NR, NMN is difficult to selectively phosphorylate in chemical synthesis. Current production methods are too costly and wasteful. as they involve lysing yeast cells and extracting NMN. As of 2016, NMN costs about $2,000 per gram(Sigma-Aldrich). An example of the relative costs associated with animal studies includes the Sinclair Lab’s experiments in which mice were given 500 mg per kilogram of body weight per day, a dose of NMN that produced dramatic reversals in aging phenotypes,resulting in an estimated cost of$$for completion of the study. (Gomes et al., 2013). For clinical studies, the cost to treat one individual would be$36,060/day for a 72.12-kg average American female and $43,320/day for an 86.64-kilogram average American male. Even with lowering the dosage to 300 mg per kilogram of bodyweight per day, the costs would still be in the tens of thousands of dollars. (Picciotto et al., 2016). With these costs, animal research with NMN is difficult to do for more than short periods of time. Long-term clinical trials with this molecule would be nearly impossible.

2. Specific Aims

Our approach to drastically reduce the cost of NMN is to genetically engineer bacteria to make excess NMN and secrete the molecule continuously. Because it is a highly-conserved simple nucleotide, the NMN found in quickly-dividing prokaryotes is identical to the NMN found in slower-growing eukaryotes (Zhou et al., 2011). By lowering expression of enzymes that inhibit NMN and upregulating activity of enzymes that increase NMN production, the bacteria can be modified to produce more NMN than they can use for themselves. In addition, modifying the pre-existing export channels in the bacteria to act as a shunt for excess NMN will allow the bacteria to further increase the NMN production without causing negative effects on the bacteria’s own metabolic function.(see figure?) Next, we aim to apply the same strategy to up regulate the production and secretion of NMN in a probiotic bacteria. Adding the same genetic modifiers for enhanced NMN production to bacteria that live in the gut would allow the molecule to become systemic as soon as it’s produced, reducing the amount of NMN needed for the desired effect. When fully operational, this system could lower the cost of an effective human NMN-based therapy from tens of thousands of dollars per day, to a scale of less than $100/month.

3. Methods

3.1. Important Genes and Genetic Circuit Overview

3.1.1. Bacterial Host 1.0: E. coli Nissle 1917

The bacterium E. coli Nissle 1917 (EcN) is being used as the bacterial strain for the first prototype. E. coli is a widely used chassis in synthetic biology, and EcN has been demonstrated to be a safe probiotic (Kruis et al., 2004). The non-pathogenic strain of E. coli does not carry pathogenic adhesion factors, does not produce any enterotoxins or cytotoxins, is not invasive, is not uropathogenic, and is rapidly killed by nonspecific defense factors of blood serum. EcN has the added benefit of increasing epithelial defensin production in its host, promoting colonic motility, and having a special lipopolysaccharide (LPS) with anti-inflammatory effects in its outer cell membrane (Sonnenborn & Schulze, 2009). EcN has also been modified for use in vaccine and pharmaceutical preparations (Ou & Yang, 2016), further demonstrating its safety and usefulness.

This bacterial strain has been confirmed to have the genes nudC, nadC, and pncB, according to analysis of the genome. We have PCR amplified nudC and nadC. The pncB from the salvage pathway will be added in via de novo synthesized DNA, mainly due to it’s size and rate of error of PCR. Confirmation of this strain’s identity has not been achieved beyond documentation from Mutafluor. Current options for identification of the strain involve 16S RNA sequencing and full genome sequencing using one of the available MinIONs.

3.1.2. Nicotinate-Nucleotide Pyrophosphorylase [carboxylating] (nadC)

This gene is involved in the de novo pathway of NAD+ synthesis, specifically the catabolism of quinolinic acid (QA). We currently have some preliminary evidence of this strain as EcN based on PCR amplification of regions from 2 plasmids that are unique to EcN. This results in a banding pattern of 3 relatively small fragments that are not present in other E coli strains. We are working toward optimizing that assay because we are getting false positives in the control E coli strain (K12).

3.1.3. NADH pyrophosphatase (nudC)

This gene is involved in the de novo pathway of NAD+ synthesis, specifically it catalyzes the hydrolysis of a broad range of dinucleotide pyrophosphates, but uniquely prefers the reduced form of NADH. NADH pyrophosphatase is involved in the cleaving of NAD+ into two NMN units.

3.1.4. Nicotinate phosphoribosyltransferase (pncB)

We identified one gene called pncB which encodes nicotinic acid phosphoribosyltransferase (NAPRTase) (Liang et al., 2013), as seen in Figure 3 (based on Zhou et al., 2011). PncB is responsible for the conversion of NA into NAMN. We selected this gene as experiments have shown that co-overexpression with another gene, nadE, increased the NAD(H) by 7-fold. This was of particular use to us as the shuttling mechanisms, among others, as mentioned previously are able to oxidise the NAD(H) into NAD+ (White and Schenk, 2012). Liang et al., 2013 co-overexpressed pncB with nadD in the E. coli CA102 strain, a variant of the E coli ?BA002 deletion strain which cannot use glucose anaerobically due to an inability to regenerate NAD+. In addition to a larger NADH/NAD+ pool, the ratio of NADH/NAD+ decreased from 0.60 to 0.12.

3.1.5. Mutant Nicotinamide Riboside Transporter PnuC (pnuC*)

One of the crucial components of the NMN-secreting microbe is the ability to transport NMN out of the cell. Grose et al., 2005 described periplasmic transporters used by Salmonella enterica for obtaining pyridine from exogenous NMN.

In the route described by Grose et al., 2005, phosphate is removed from NMN in the periplasm by acid phosphatase (AphA), and the produced nicotinamide ribonucleoside (NmR) enters the cell via the nicotinamide riboside transporter (PnuC). Internal NmR is then converted back to NMN by the NmR kinase activity of NadR. In pnuC* transporter mutants, NMN can be imported intact can therefore grow on lower levels of NMN. If the protein were modified so the intermembrane sections of the protein were reversed, this could be adapted to secrete NMN into the periplasm from inside the cell.

Since mutating the bacterium’s only gene for this transporter would disrupt the normal function of the cell and be detrimental to viability, a transgene version of this transporter will be inserted. This also offers the opportunity to choose from homologs of the PnuC transporter that may exist in a wide variety of organisms.

According to Uniprot, E. coli also has a PnuC transporter. The E. coli PnuC transporter is a multi-pass membrane protein with transmembrane domains making up residues 22-42, 49-68, 72-89, 110-127, 158-177, and 184-206.

PnuC is also repressed by NadR (Grose et al., 2005). Should regulation of the mutant transporter levels by an inducible promoter fail, this may be a promising route with which to manipulate the activity of the transporters.

3.1.6. Mouse Nicotinamide Phosphoribosyltransferase (mNampt)

We also have an opportunity to incorporate eukaryotic genes into the bacterium. Mammalian cells have nicotinamide phosphoribosyltransferase, or Nampt, which converts to nicotinamide to nicotinamide mononucleotide. Nampt is a feature of the metabolism of only vertebrates, all of which have skeletal muscle and therefore have cells that need to release a lot of energy quickly (Kim et al., 2014). The NAMPT gene does not have any known homologs in bacteria, fungi, or non-vertebrates. This will be a highly effective addition to the bacterial pathway.

For acquiring this gene, Abnova sells bioactive recombinant mouse Nampt in an E. coli vector. This catalytic activity does not require co-factors other than initial Nicotinamide D-ribonucleotide & diphosphate reactants.

3.1.7. Nampt-Prs1 Fusion Protein

Fusing Prs1 to Nampt ensures that the PRPP, one of the substrates for Nampt, is locally available at high concentration.

3.1.8. Nampt-Prs1 Fusion Protein

Pathway control

3.1.9. Plasmids for Delivery Strategy

The genes of interest will be modified in different combinations when the E. coli Nissle 1917 bacteria are transformed. The metabolome of the modified EcN will be measured and compared with bacteria of the same strain without the plasmid, as well as the unmodified reference strain E. coli K-12 MG1655 CGSC#6300.

Given these experimental setups, we would be able to combinatorially verify the contribution of each additional gene to the change in NMN levels. With this many transformations, the choice of plasmid will be especially important.

We are currently putting amplicons into a linearized TOPO vector, just a simple TA cloning. We still need to determine a better expression vector, but we can pop it out and move it around using restriction enzymes from this plasmid. Alternatively, we can add a restriction site onto the end of the primer and use that in a directed cloning.

Two of the more promising empty vectors for use are Addgene’s pBAD LIC Cloning vector (plasmid #37501) and pET15 (plasmid #26092).

Both of these plasmids feature ampicillin resistance selection markers. These markers are compatible with both ampicillin-laced media and carbenicillin-laced media. Carbenicillin will be favored since it does not allow as many satellite colonies to form in areas where β-lactamase has rendered antibiotic harmless. Carbenicillin is also far more heat stable than ampicillin, and is less toxic to humans than similarly accessible antibiotics like kanamycin and erythromycin. Due to it’s compatibility with E. coli nissle 1917, we are looking into the pQE-80L as a vector.

The pQE-80L plasmid also has variants pQE-81L and pQE-82L. All of these contain His-tags, but for our purposes we do not need to purify proteins so this would be redundant.

3.1.9. Primers used in this Project

Many DNA primers will be used in this project for cloning and amplification. The Muta Rev, Muta9-For, Muta8-Rev, Muta7-For, Muta6-Rev, and Muta5-For primers are based on the E. coli Nissle 1917 characterization PCR reactions described in Blum-Oehler et al., 2002.

The primers used in this project will be obtained from Integrated DNA Technologies.

3.2. Transformation Strategy

3.2.1. Electrical Transformation

E. coli Nissle 1917 is best transformed with Electroporation. Our electroporation protocol is adapted from the New England Biolabs protocol for making electrocompetent cells, and was optimized for use with our labs electroporators (👾). This also has the added benefit of being compatible with automated directed evolution methods like MAGE.

3.2.2. Heat Shock Transformation

Should Electroporation fail, chemical transformation will be used. As of January 2017, Heat Shock transformation has thus far been the most successful method for transforming bacteria. BLANK has been optimizing a heat shock protocol that is ideal for use in our space (👾).

3.3. Media

3.3.1 Transformation

Super Optimized Broth (SOB) will be used, if available, to increase transformation efficiency. Media with carbenicillin will be used for bacteria with ampicillin resistance plasmids. Carbenicillin is a chemical analog to ampicillin with two advantages over ampicillin: one, it is more stable at 37°C (making it better for both incubating bacteria and being stored long term in a -20°C or -80°C freezer); two, it is more resistant to degradation (reducing the number of satellite colonies of non-transformed bacteria living on small sections of agar that have been cleared of antibiotic by the truly resistant ones). Kanamycin will also be used as a selection strategy.

3.3.2. Visualization

According to Sebastian S. Cocioba, a research affiliate at the MIT media lab, activated charcoal can be added to LB agar at a concentration of 10 grams/liter. The result is media that is jet black. This media type could be useful in visualizing the colonies grown on it, especially as the E. coli Nissle 1917 has a slightly different morphology than E. coli strains like DH5α. There is also anecdotal evidence that bacteria also grow better on media containing activated charcoal than without.

3.3.3. Metabolite-Specific Yield-Optimization

Media with niacin could be used to make sure the bacterium uses. Niacin is an upstream precursor to the molecules in the NAD+ pathway. We use LB as a general growth medium for E. coli and can include niacin to assist in driving the salvage pathway for NAD+ production, potentially increasing NMN quantities. Liang et al., 2013 found that when Sorbitol was used instead of glucose as a carbon source, NAD+ levels increased.

3.3.4. Considerations for Universal Media

Once the genetic circuits have been optimized in EcN, and are being moved to probiotic species of bacteria, universal media may be required due to more stringent growth prerequisites. The advantage of using bacteria like these as chassis is the promise of creating probiotics that take more permanent holds in the gut microbiome through biofilms. Universal media protocols are being developed by labs researching probiotics, as many species in the human microbiome are not easily culturable with traditional LB media. Arguably, the primary reason for the resistance of bacteria to in vitro cultivation (in isolation) is a dependence on other bacteria and on chemical signals within mixed communities (Vartoukian et al., 2010; Stewart et al., 2012). This may be particularly relevant to bacteria inhabiting biofilm-type communities, such as dental plaque, where metabolic cooperation and intercellular signaling networks are widespread (Mihai et al., 2015).

3.4. Measurement

3.4.1. Liquid Chromatography- Mass Spectrometry

Mass Spectrometry is the best method for quantifying NMN levels directly. Many protocols for quantifying metabolites related to NAD+ involve the use of Liquid Chromatography-Mass Spectrometry (LC-MS) (Trammell & Brenner, 2013). Part of the protocol in Trammell & Brenner, 2013, which was tested out on eukaryotic cells, involves using a triple quadrupole (QQQ) trap to separate out the noise from all the other compounds within a cell. Without the use of the QQQ trap, identifying the NMN and other desired metabolites from the data will require a lot more. Regarding the fixing of the samples many protocols, such as that of the Boston University Chemical Instrumentation Center (CIC) cite the use of boiled buffered ethanol as the best reagent to use. The Boston University CIC is by far the most economical option for Mass Spectrometry. Before Ginkgo Bioworks had its own Mass Spec Facility, this was the resource they used for measuring molecules. The Beth Israel Deaconess Medical Center has a Mass Spectrometry facility that offers metabolomics services. They are also the same facility used by the Sinclair and Haigis Labs for their NAD+ metabolomics experiments.

3.4.2. Indirect Measurement with Plate-Reader Kit

There are multiple options for measurement of NMN with fluorometric assays (Zamporlini et al., 2014). The Promega Glo NAD+/NADH assay kit will be marginally cheaper and much faster to run than Mass Spectrometry. The genes of interest in the experiments would most likely also need to be analyzed for expression levels via cDNA analysis.

3.4.3. Trade-offs and Considerations between Direct and Indirect Measurement

To directly respond to your email; the kits pay for many samples, probably all the samples you’ll need to run. Running a sample down mass spec will cost you at least $50/sample. Because the genes that we’ll clone or synthesize haven’t been tested in our cell line, we will need to run triplicates of the assay at varying time points, of different dilutions of each of your experiments.

3.5. Strain Optimization and Directed Evolution

3.5.1. Phage-assisted continuous evolution (PACE)

PACE is being considered for the directed evolution component. PACE is a single-pot directed evolution setup that allows for the optimization of enzymes placed within phage vectors that use E. coli as a host (Esvelt et al., 2011). Since we are using an E. coli strain for our initial prototype, this may be a feasible method for optimizing single enzymes like NadD for greater catalytic efficiency.

3.5.2. Multiplex automated Genome Engineering (MAGE)

MAGE is an automated directed evolution platform that cycles cultures through mutation and selection. MAGE does require the construction of biosensors prior to the automation process. This particular method of metabolic engineering, and its associated sensor design, are described in Taylor et al., 2016, Raman et al., 2014, and Rogers et al., 2016. Feng et al., 2015 focuses on sensor design in eukaryotes, although some of the principles might still apply to prokaryotes like the ones we will be using.

3.6. Purification Options

3.6.1. Centrifugation

Simple Centrifugation-based methods would clearly be the most cost-effective. We already have both a bench-top centrifuge (Eppendorf 5415 C) for 2 mL individual volumes (18 × 1.5-2.0 mL wells, max speed of 14,000 rpm/15,996 g) and a larger centrifuge (Eppendorf 5810 R) for 50mL individual volumes (4 × 50 mL wells × 2, max speed of 14,000 rpm/35,280 g)

3.6.2. Protein purification gel

Nicotinamide mononucleotide is not a peptide, or amino-acid-based macromolecule. Unless NMN is bound to a protein, which would be metabolically straining and inefficient, a polyacrylamide gel would therefore be insufficient for separating the NMN from the lysate.

3.6.3. High Performance Liquid Chromatography (HPLC)

Liquid chromatography is commonly used for the purification of NMN, as well as for verification of its purity. For our purposes, a low pressure column and old machine would suffice (as long as it includes contact closure inputs). Based on correspondence with HPLC machine operators, “The uglier the machine, the better. The newer stuff just breaks.” Several sources have also recommended using an old machine for the sake of controlling it with an Arduino. Detection would involve looking for cyclic rings in the column using fluorescence. High UV, either 214 nm or 280 nm could also be used. The longer the flow cell path, the stronger signal we will get. As for acquiring an HPLC machine, we could potentially get one from a lab that doesn’t want to use it anymore. For example, we could search for a Waters 600 Quat Pump and Waters 600 controller (LCD), a Waters 717 plus autosampler, a Waters 2887 Dual Lambda Absorbance Detector, and an In-Line Degasser ISA Card.

3.7. Computer Simulations

3.7.1. Sources of Enzymatic information

It will not be practical to experimentally verify the enzymatic rates and constants on our own at this moment.

Uniprot is thus far the most reliable source of enzymatic information on the NAD-dependent pathways.

In 2012, Karr et al., 2012 claimed to have successfully created a whole-cell simulation of M. gentiatlium (much of the enzymatic data was filled in with information from E. coli though, so it’s not an entirely faithful simulation). The simulation does have space for NAD+ and NADH though, but no other molecular intermediates for that pathway. It may still be a useful resource for constructing the differential equations for the pathway. Many of the papers cited in the references for the work will also be useful.

3.7.2. Equations and Terms

Many of the enzymatic activities in the pathway can be simulated with Ordinary Differential Equations (ODEs).

The 2016 Northeastern University iGEM team provided some information on simulating NAD+-dependent enzymes in E. coli. While none of the precursors to NAD+ were explicitly shown in their model, it may be a useful jumping off point for simulating the levels of the metabolites. Rutkis et al., 2013 may also be useful as a guide for putting together the model.

Given the limited nature of deterministic equations like this simulating probabilistic systems like cells, it may also be worthwhile to construct stochastic equation alternatives for each of the molecules in the pathway.

3.7.3. Machine Learning Techniques

The problem with many of the pathway information sources is that they are incomplete. This problem as addressed by Daniels & Nemenman, 2014. They used two softwares, Sir Issac and Eureqa, to piece together the missing information in cell regulatory dynamics. This was demonstrated by constructing a coarse-grained simulation of yeast glycolysis, which started with more than half of the necessary information being missing.

This is a tentative outline of the timeline for the project. It is loosely based on the project timeline for the Open Insulin project.

Figure 4: Visual overview of prototyping strategy for project agetuneup. The project will start out by using wild-type E. coli Nissle 1917 (A) as a chassis. This bacterium will start out having completely unmodified enzyme kinetics and metabolite regulation. The next stage (B) will involve upregulating the amount of NMN in the cell, with levels of upstream molecules like NR and downstream molecules like NAD+ remaining as close to normal as possible. The next stage (C) will involve transporting this excess NMN into the extracellular space. From here, the project can diverge in two directions. The first (D) is the reapplication of the transformation strategy to probiotic bacteria like B. longum, which could take more permanent residence in the gut and secrete NMN for systemic absorption. The second (E) is the application of the transformation strategy to quickly-growing bacteria like V. natriegens. This would allow more rapid production of NMN in a bioreactor, for use in non-probiotic applications of the molecule. V. natriegens also has type I, II, and III extracellular transport systems that could be utilized.

4. Budget

We have concluded that $50,000 is needed to effectively initiate Stage 1 of the project. In Stage 1, the team will introduce multiple DNA constructs into E. coli Nissle 1917 bacteria, which will cause changes in NAD+ and NMN levels. We are trying several different strategies for modifying the metabolism of these molecules. The funds will also pay for the assay kits and enzymes used to measure levels of NMN, as well as the use of mass spectroscopy facilities at Boston University for verifying the results of these kits. At this stage in the project, the purification of NMN will be feasible through relatively simple and inexpensive centrifugation-based methods. Money raised past this minimum will be used to create further mutations in the bacteria to improve NMN production, apply machine learning to make predictive models of the fitness landscape, and test the design in other bacteria species that are either better suited for large scale production (such as the V. natriegens described in Lee et al., 2016), or better suited for probiotic use (such as the B. longum described in Hoy-Schulz et al., 2016) This project relies entirely on volunteer labor. The cost of external facilities is currently supported by Sprout&co. Any additional funds will go toward the next stage of the project, using phage-based recombineering to increase production of NMN, (Esvelt et al., 2011; Badran et al., 2015) designing export channels in the cell membranes, applying machine learning techniques to the screening of the bacteria we produce, and accelerating our progress by hiring commercial services to supplement our volunteer labor.

5. Timeline

5.1. Overview

Stage 0: Creating MVP for fundraising (current) Get plasmid with NadC, NudC & PncB or Get plasmid with mNampt Put plasmid in bacteria Get plate reader (used) Get indirect measurements of NMN Get necessary documents in order for further fundraising Budget: $3000 Time Frame: 3 months Stage 1: Proof of Principle experiments

• Designing, Synthesizing, and Testing constructs
• Making the NMN in excess with bacteria
• Setting up recombineering workflow Budget: $50K (to be raised over 2 months through crowdfunding) Time Frame: 6 to 12 months Stage 2: Improvements needed to meet research needs
• Optimize bacteria productivity with further metabolic engineering
• Set up collaborations with labs investing aging and age-related chronic diseases
• Supply enough NMN for C. elegans testing
• Supply enough NMN for Mouse testing
• Check for effects on cancers (may need to compensate with substance like spermidine) Budget: $300K Time Frame: ~ 1 year Stage 3: Get ready for Human Treatments
• Scale up
• Beginning Human trials
• Make Deal with FDA Budget: $3-5M? Time Frame: ~ 3 years

This is a tentative outline of the timeline for the project. It is loosely based on the project timeline for the Open Insulin project.

5.2. Stage 0: Creating MVP for fundraising

Get plasmid with NadC, NudC & PncB Or Get plasmid with mNampt

Put plasmid in bacteria Get plate reader (used) Get indirect measurements of NMN

For the time being, we are still on the stage of generating a minimum viable product. This is not intended to provide the functions of the end probiotic, but rather to demonstrate that the team can modify the bacteria to make more NMN, as well as to measure it. This MVP would not include any of the pores in the membrane that would be used to transport the molecule outside the molecule. For the purposes of crowdfunding or attracting larger cash cow investors, a lyophilizer could be used to dehydrate cultures of the bacteria and place them in a vegetable-based capsule (like one from a health food store that costs about $0.12). This capsule would in no way be intended to be consumed, but would provide a visual of what we hope the end product of this project would look like. Functionally this would be more similar to the props used

5.3. Stage 1: Designing, Synthesizing, and Testing constructs

Making the NMN in excess with bacteria Setting up recombineering workflow Budget: $50K (to be raised over 2 months through crowdfunding) Time Frame: 6 to 12 months

5.4. Stage 2: Improvements needed to meet research needs

Optimize bacteria productivity with further metabolic engineering Set up collaborations with labs investing aging and age-related chronic diseases Supply enough NMN for C. elegans testing Supply enough NMN for Mouse testing Check for effects on cancers (may need to compensate with substance like spermidine) Budget: $300K Time Frame: ~ 1 year 5.5. Stage 3: Get ready for Human Treatments Scale up Beginning Human trials Make Deal with FDA Budget:$1-5M? Time Frame: ~ 3 to 5 years

6. Project Team Bio (REDACTED)

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7. Frequently Asked Questions (FAQ)

7.1. “Wait a minute. Are you talking about a genetically modified organism (GMO)?”

Yes, we are genetically modifying E. coli for this project. Since we are keeping safety in mind, we are using an E. coli strain that is often sold as a probiotic supplement and is not pathogenic. (Kruis et al., 2004) For the culturing of bacteria to harvest large amounts of NMN, these bacteria will be in a contained environment and not come into contact with humans. The later stages of the project aimed at creating the full working probiotic would incorporate bacteria that are already approved by the FDA. While the new strains would require re-approval, this would come after the safety testing with the NMN itself on a much larger scale. The capacity for these strains to cause negative health effects would be fully investigated, though we expect them to be negligible.

7.2. “Is E. coli being used as the host organism mainly because it’s already widely used? Why not S. cerevisiae or B. subtilis?”

That is one reason E. coli is being used, but there are a lot of other reasons as well. The specific E. coli strain we are using at the moment, E. coli Nissle 1917, is perfectly suited to act as both a prototype for the production as well as a proof of concept for the probiotic. (Kruis et al., 2004) Certain bacteria may be better for sitting in a bioreactor and producing NMN, and some may be better suited for the task of producing this molecule in a mammalian GI tract, but this E. coli strain we’re using is the perfect system to produce a minimum viable product. S. cerevisiae is a current source of the $1,000-$2000/gram NMN. The S. cerevisiae is grown in large amounts and then lysed to obtain the NMN inside. Under ideal conditions, most S. cerevisiae cultures have a population-doubling time of approximately 90-140 minutes. E. coli takes about 15-20 minutes (not counting special, rapidly dividing strains). Prokaryotes are easier to engineer and easier to subject to directed evolution. The E. coli NAD+ pathway, at least in our experience, seems to be a lot easier to manipulate without killing the organism than the pathway in yeast, so there is a malleability advantage. Plus, one of our goals is to create a synthetic probiotic that can produce NMN. This would most easily be achieved by modifying a prokaryotic NMN-supply system. Engineering fungi for living in the GI tract would be a far more challenging and dangerous feat. B. subtilis is gram positive, meaning it has a thicker peptidoglycan wall. For engineering the export channels for this molecule, we are starting with a gram-negative species for the sake of engineering simplicity. Something like B. subtilis would probably require something like osmotic shock to get the NMN out. While in the future we would work on the best export channel for whatever bacterial species we are using for a probiotic, for the current short term we are focusing on the simplest path to creating a shunt for NMN. Once we achieve that, there will be greater room for the bacteria to increase NMN production since the shunt will make sure NMN levels don’t get high enough in the cell to cause problems.

7.3. “What is the reasoning behind the feasibility of this $100/month cost?”

E. coli cells are typically rod-shaped, and are about 2.0 μm long and 0.25–1.0 μm in diameter, with a cell volume of 0.6–0.7 μm3. For the production of metabolites optimal production would happen during log phase when the optical density at 600 nm (OD600) is highest before growth slows down. Sezonov et al., ‎2007 determined that this happened at an OD600 of 0.3 in Luria-Bertani Broth. From this, one can calculate the amount of E. coli at this highly metabolically-active time point, how much it takes to raise them, harvest a product from them, and determine how much NMN each cell needs to produce to meet the $100/month cost Part of the estimate for the$100/month cost comes from the vision of a synthetic probiotic that acts as an internal factory for NMN. The price would be on the bacteria, not so much the NMN itself. The closest thing physically on the market to this would be lyophilized E. coli containing recombinant mouse Nampt. These are sold by the Taiwanese company Abnova in 10 µg units that cost USD $259 each (not including the cost of shipping from Taiwan). This product is physically similar to what we want, both are bacteria containing NAD+ metabolism modifying genes. The Abnova bacterium is a research tool, however, and would likely have drastically different supply and demand curves. As a bacterium, it could probably be grown in quantities far larger than 10 µg. With that in mind, plus the fact that a successful enough anti-aging bacteria could conceivably be subsidized by a nonprofit or government grant, the$100/month goal is definitely within reach.

7.4. “If you’re trying to go with a probiotic, why not modify bacteria like Lactobacillus acidophilus or Clostridium?”

Lactobacillus acidophilus is the bacterium of choice for many synthetic probiotic projects. In our experience however, the majority of strains have been grown in laboratory environments for so long that they lack the salvage pathways that we wish to manipulate. Clostridium has shown to be very useful in microbiome applications, mainly due to a. However, a wide-sweeping patent was recently filed for by Vendanta Biosciences which broadly covers applications of this genus of bacteria. It would be safer, as well as less legally troublesome, to go with another probiotic species.

7.5. “What are some of the risks involved? What do we know about possible negative biological or toxological effects?”

We are confident we will be able to see this project to completion. The risks involved fall into three categories. The first category is transformation. We have been very careful about the plans for genetic modification so we can make the desired bacteria. That being said, manipulating biology isn’t always as predictable as programming a computer and version 1.0 of the bacterium might not be a fire-hose of NMN that we would all like. We are addressing this risk by planning directed evolution experiments. This will allow us to improve the microbe through means that are beyond human design capabilities. The more money we raise, the more we can refine our designs, the more directed evolution experiments we can run, and the stronger the effect we will get. The second category is the biological effects of the NMN. NMN was chosen for this molecule because it has had some very promising results in early mouse testing. There have not been any longitudinal lifespan experiments on these animals yet, so it has not been verified that it can slow aging, or if so how it’s ability compares to compounds like rapamycin or metformin. Given the results of NMN reducing biomarkers of aging in the short term, this poses a very small risk to the success of the project. The third category is regulation. This is still a new field, and it is possible that more restrictive regulations would be implemented during the project that will require us to get a permit which would delay the release of the bacteria, or possibly block the release of the probiotic phase altogether. Even then, the bacteria we are making would still have use as a manufacturing tool for NMN, which would not require the same kind of approval as a medical device. There would still be options for the project.

7.6. “Besides general aging, which specific diseases do you think this would be most effective against?”

We mentioned a list of diseases and health conditions that defective sirtuin function plays a part in Hall et al., 2013. Since NMN is so expensive, there hasn’t been a comprehensive screen of the molecule’s effects on different cell types. Given what we have seen so far regarding the molecule’s effects on aging pathways and specific diseases, it bears many similarities to exercise in terms of effects on health (Kolati et al., 2010; White & Schenk, 2012). Diabetes would be our best bet regarding which health condition the molecule would be most effective against, simply due to the amount of documentation of overlap between diabetes-related metabolic pathways and pathways containing NAD-dependent proteins (Yoshino et al., 2011). This would be followed by neurodegenerative diseases, which we guess based on the increasing interest in the bioenergetic effects on conditions like Alzheimer’s and Parkinson’s. (Wang et al., 2015; Wilkins et al., 2014; Mattson et al., 2014; Ying, 2007) Just as with NAD+, the mitochondria are a large area of focus for such diseases. (Yao and Brinton, 2011; Van De Weijer et al., 2015) NAD-synthesizing enzymes, like NMNATs, that maintain the proper flux of NAD in specific tissues are also a potential target. (Ali et al., 2013; Conforti et al., 2014). One isoform of this, NMNAT2, is highly expressed in the mammalian brain (Mayer et al., 2010; Orsomando et al., 2012). nmnat2 mRNA levels are reduced in Parkinson, Huntington, and Alzheimer diseases (AD), as well as in tauopathies and proteinopathies (Liang et al., 2007; Liang et al., 2008; Van Deerlin et al., 2010; Lesnick et al., 2007; Moran et al., 2006; Hodges et al., 2006; Ali et al., 2016). Supplying cells deficient in NMNATs with the substrate for their activity might reduce the disease burden for a litany of neurodegenerative diseases. Conditions like Diabetes and neurodegenerative disease would be followed by arterial dysfunction and other cardiovascular diseases in terms of most likely effective target diseases. Gomes et al., 2013 showed that NMN supplementation simulated hypoxia in muscle cells. Actual systemic hypoxemia inhibits aerobic respiration, thus reducing damage from reactive oxygen species. Nakada et al., 2016 demonstrated that in adult mice gradually exposed to severe systemic hypoxemia (where inspired oxygen is gradually decreased by 1% and maintained at 7% for two weeks) do in fact have inhibited oxidative metabolism, decreased ROS production, decreased oxidative DNA damage, and reactivation of cardiomyocyte mitosis. It is unknown where cancer would fall on this ranking of responsiveness to a sirtuin-activating compound. SIRT6 has protective effects against cancer, while SIRT7 has aggravative effects (Yang et al., 2009; Barber et al., 2012). With sirtuins like SIRT1, SIRT2, and SIRT3, there are disparities between cell types in terms of whether they are protective or aggravative (Fang & Nicholl et al., 2011; Lain et al., 2008; Haigis et al., 2011). SIRT5 has seen little characterization by comparison, although NMN could undoubtedly be useful in remedying this. NMN could be useful in cancer prevention more than treatment due it it’s ability to affect protein-folding. Zhou et al., 2015 demonstrated rescuing of protein misfolding in neurons by replenishing NAD+ levels. There is mounting evidence that misfolding of proteins, such as p53, can contribute to cancer development and metastasis (Xu et al., 2011).

7.7. “You gave some vague details about the current system, which is using S. cerevisiae for producing NMN. How exactly is that done?”

The S. cerevisiae system involves transfecting the cells with mouse NAMPT to convert nicotinamide riboside directly to nicotinamide mononucleotide. Ours uses similar principles of upregulating the upstream enzymes of NMN production and downregulating the downstream enzymes.

7.8. “Can you give more details about the stretch goals?”

Much of the stretch goals involve expansion of the efforts for directed evolution. This part of the problem is relatively straightforward, and its success is more predictably described with a linear function showing success probability as a function of funds. Beyond just running the experiments for longer, it will allow us to use both selection and screening to improve the strains.
One of the stretch goals would also be to pay for more sequencing so we would have more genomic data for the selection process. Once we do that, we plan on analyzing these genomic data using software like Galaxy and Eureqa. The first of these is a browser-based software that creates customizable bioinformatics workflows. (Afgan et al., 2016; Zelewska et al., 2016) The second is a data-mining software that searches the entirety of mathematical space for equations and patterns that fit the data it has been given. (Schmidt & Lipson, 2013; Weidlich et al., 2013) Both goals would be made possible if the campaign went $15,000 past its main goal. Should we make an additional$25,000, we would work on setting up more of the equipment for C. elegans testing. We have access to both the source code (🐙), the bill of parts (🐙), and the instructions for construction and use (🐙) of a C. elegans Lifespan Machine (Stroustrup et al., 2013).

7.9. “Is this something the FDA would approve of?”

With the NMN molecule itself, and providing the molecule to researchers for use with model organisms, there would most likely be need for extensive regulatory approval than for something like a drug or medical device (save for experiments involving mice, which would involve putting together standardized protocols with IACUC). Should the verifications of NMN’s efficacy and safety be successful, we would work with the FDA on setting up human trials and getting it approved for human use. Since NMN is a naturally-occurring molecule found in most cells, the testing would follow the FDA’s guidelines for safety testing for metabolites. Approval of the synthetic microbes would be a separate matter. The FDA does not have a well-defined process for approval of synthetic commensal microbes, although there are many probiotics approved for human consumption today, such as Bifidobacterium, so it is far from impossible. In many cases, probiotics are classified by their intended use. An NMN-secreting probiotic might fall into the category of a biologic, provided sufficient characterization of the microorganism. Based on our correspondence with Carrie McMahon, Ph.D from the FDA’s Office of Food Additive Safety, the approval of this aspect of the probiotic would be a task for the Center for Biologic Evaluation & Research. The efforts to investigate the properties of the NMN compound itself by the scientific community should make it much easier to get an NMN-secreting microbe approved. Getting FDA approval for use of something like this in humans is still a far off. It is highly possible that by the time this project does get to this stage, the FDA will have reacted to mounting industry pressure to put together a more concrete approval process. It could also become more stringent. Even if the microbe itself is not approved for use as a probiotic, there is still the option of using the microbe as a source of NMN to be distilled and consumed on its own.

7.10. “Who are the mentors and advisors on this project?”

REDACTED

7.11. “What is the difference between the NMN and the NR supplements that are for sale on the internet?”

NR (nicotinamide riboside) is another precursor to NMN. The logic behind using NR is similar to the reasoning for using NMN, although NR is further upstream in the pathway. This means there are more opportunities to be turned into something else other than NAD+, meaning it would be less potent. NMN could also have fewer side effects compared to NR. There are many companies selling supplements purported to contain NR. Some of these NR supplements may also be sold with compounds like resveratrol or pterostilbene. In the overwhelming majority of these cases, the compounds are not in large enough quantities to have any visible physiological effect. To replicate the mouse experiments with NR would require a high dosage. If the supplement capsules contained as much NR as was claimed by the sellers, one would most likely need to consume an entire bottle of the supplements in one sitting to have the desired effect. Even then, the supplements market is subject to very little regulation. Some of the brands of NR supplement may contain less than the stated amount on the bottle, or perhaps none at all. They are most likely acting as placebos. In addition to focusing on NMN, the more promising NAD-precursor than NR, our goal is to supply biomedical researchers with this hard-to-obtain compound. Should the resulting research show promising results, we would then work with organizations focusing on specific diseases to find the optimal medical usage for NMN. We are not going to be selling supplements with unverified effects and ingredients. The specific company selling these supplements, Elysium Health, seems to have also copied their business model from Tim Ferriss’s “Brain Quicken” company. The founders appear to have copied the model outlined in “the 4-hour workweek”, including the section on passing yourself off as an expert. They might not be the most trustworthy source of anti-aging regimens.

7.12. “Are there any side effects to NMN?”

NMN supplementation has no known side effects because the barrier for such research has been very high. The closest thing we have as a reference is toxicology studies done on nicotinamide riboside. The journal Human and Experimental Toxicology (HET) published a study in 2016 which looked at nicotinamide riboside safety. (Conze et al., 2016) The paper mentions the first side effects being visible at 1000 mg/kg/day. They did not state the exact overdose symptoms, but the liver, kidney, ovaries, and testes were the “target organs” that they were looking for toxicity in. Presumably the side effects were related to one or more of those. With toxicity tests, the liver is by far the most commonly affected one in supplement overdoses. There was no observed genotoxicity. Frederick et al., 2015 observed that despite being involved in energy metabolism, increased oxidative stress is not a side effect of higher NAD+ levels. By extension it is probably not a symptom of NMN either. Again, these side effects are for a molecule closely-related to NMN, not NMN itself. One of the things we are hoping to do with this project is allow researchers asking questions like this to do investigations they want.

7.13. “How is this project being funded currently?”

REDACTED

7.14. “What are some other options for funding?”

REDACTED

7.15. “Why not make pure NAD+ instead of NMN?”

NAD+ is a charged molecule that often has a more difficult time getting into cells than NMN. There is also mounting evidence that NAD+ is to some degree involved in extracellular signaling, such as in signaling of tissue damage (Ido et al., 2001; Billington et al., 2006; Koch-Nolte et al., 2011). Use of NMN would be less likely to cause undesirable disruption to the endocrine system.

7.16. “I heard of another company, Ageria Bio, that was also working on efforts to create probiotics that fight aging. Is this project similar? What is the difference between this project and what they’re doing?”

There are also many differences between the management models for the project. Ageria Bio is a private company that is focusing on probiotics from Yogurt. Our project has a few differences Ageria Bio’s. The FDA is far less stringent than the European Union’s food safety commission. It is also possible that it may get even less stringent in the upcoming political climate. The founding team is also divided between Ohio, USA, and Austria. Ageria Bio is also working to develop bacteria that secrete molecules already made in large, economical amounts. NMN on the other hand is comparable in price/weight to plutonium. Our microbe would be useful to researchers even without the probiotic chassis. It is currently unknown how active Ageria Bio’s project is. Their last update on the Company’s Blog was more than a year ago. This is a stark contrast against the company’s previous monthly reports and posts. They had also switched away from S-adenosylmethionine to spermidine (the latter would likely pose the problem of making probiotic yogurt smell rancid even when pasteurized). They have also switched away from their previous Lactobacillus model, and at least one of the founders is involved with a new and unrelated startup.

7.17. “For fighting aging by targeting this specific pathway, wouldn’t a drug that upregulates one of the enzymes be more effective?”

Calico has licensed experimental drug compounds P7C3 analogs involved in enhancing the activity of the enzyme nicotinamide phosphoribosyltransferase, which plays a role in NAD+ biosynthesis. P7C3 compounds have previously been shown in a number of publications to be beneficial in animal models for age-related neurodegeneration (Cain, 2014; Wang et al., 2014). The partnership involves upfront and milestone payments for development of the compounds into therapeutics. The main assumption behind the use of small molecules is that if a particular enzyme in the pathway is the rate-limiting step, the small molecule can fix that. Introducing large amounts of a precursor molecule can act independently of rate-limiting enzymes, even moreso if the precursor is one step upstream of the desired product (NMN being one step removed from NAD+). In this sense, our system of supplying outside precursors is superior. Small molecules and supplements are also not mutually exclusive to small molecules. It is entirely possible that they could be combined to have a synergistic effect. This would probably be limited to a small subset of drugs though, since exogenous NMN is so close to NAD+ in the network.

7.18. “How applicable would this project be to space travel? What possible directions could the project go in after achieving the aging-based goals?”

Space travel is definitely an area of interest to many of our members. Since sirtuins repair the epigenome, which undergoes morphological changes upon the fixing of radiation damage, this would be an exciting potential application of increased NMN supplementation. General radiation sickness is another possible problem the bacterium could be modified to address. It I highly probable that the 21st century will see some kind of nuclear attack. In the resulting humanitarian crisis, there will be a huge need for effective treatments for radiation poisoning. The use of microbes themselves may be dangerous to use on someone with a compromised immune system, although this production method would be useful for the creation of therapies that include NMN as ingredients.

7.19. “Are you guys really going to stick with the name ‘Project AgeTuneUp’?”

It’s true, Project AgeTuneUp is not the most inspired name. At some point the project may be given a different name, but that would probably only happen if the project was ready to be spun out into a distinct organization (either for-profit or non-profit). It would most likely be difficult after this point, and unnecessary before this point, just like with Symbiotic renaming itself to Indigo Agriculture. We have been mulling over the name “Kalpa”, a term used to refer to an impossibly long time period in Buddhism and Hinduism. Given that our project is aimed at aging, a name like Kalpa (either for the product itself or the distinct organization distributing it) might seem appropriate. We have yet to see if there are any existing trademarks on that name, although renaming the project is of minimal priority at this time compared to all of the other technical and financial details.

7.20. “How are you going to apply rapid prototyping to synthetic biology when such systems often require long periods of time for reactions to occur?”

REDACTED

7.21. “What is being considered in terms of options for exporting NMN out of the cell?”

Our current plan is to use a modified version of the PnuC transporter that is mutated to lose its catalytic activity. There is already a large body of research That being said, it is entirely possible that this transport strategy will not work. One option is to design a “symporter” from scratch. The 2016 University of Goettingen iGEM team designed a synthetic Vitamin B-12 synporter as a concept for increasing industrial production of vitamin B12 without the need for cell lysis. Vitamin B12 contains some common groups as nicotinamide mononucleotide, but the most glaring difference between the two molecules is the presence of a large cobalt-containing corrin ring in the vitamin B12. Another option is to modify the porins of the cells, the channels used for transport of water, to be able to transport nicotinamide mononucleotide. This would in principle be similar to a project undertaken by the 2016 UC Louvain iGEM team. Both of these approaches would require the use of protein domains that can bind to NMN without influencing its structure. It might be possible to engineer something like this with a tuneable two-hybrid system, like that used by the 2016 Bielefeld iGEM team’s “Evobodies” project. The majority of these alternative plans involve concepts taken from iGEM teams. As such, there probably would be some legal issues to work through before a product incorporating features like that could be sold or developed.

7.22. “Aren’t you guys concerned about intellectual property or someone stealing the idea?”

REDACTED

7.23. “How can NMN be a better option for secretion if the absorption of both NR and NMN into cells are both dictated by the NRK1 protein in mammals?”

This question is most likely referring to the recent paper by Charles Brenner’s Lab (Ratajczak et al., 2016) tying the absorption rates of NMN and NR to the function of the mammalian NRK1 protein. If this is the case, there is a possibility that NMN would be more absorbable by the microvilli in the intestine or Charles Brenner is also an advisor to ChromaDex, which sells NR to Elysium Health. It is unsure whether that conflict of interest affected that research.

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Cited as:

@article{mcateer2015devoag,
    title = "Directed evolution for aging",
    author = "McAteer, Matthew",
    journal = "matthewmcateer.me",
    year = "2015",
    url = "https://matthewmcateer.me/blog/directed-evolution-for-aging/"
}

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