Tome Biosciences investment analysis

December 13, 2023

This is not investment advice. We used AI and automated software tools for most of this research. A human formatted the charts based on data / analysis from the software, prompted the AI to do some editing, and did some light manual editing. We did some fact checking but cannot guarantee the accuracy of everything in the article. We do not have a position in or a relationship with the company.

Overview

TOME Biosciences has developed a technology platform for Programmable Genomic Integration (PGI), enabling the precise insertion of any size DNA sequence into specific genomic locations. This platform, licensed from MIT, combines CRISPR/Cas9's site-specificity with enzymes for inserting DNA, including entire genes, without double-strand DNA breaks.

The key component of this technology is the integrase-mediated PGI (I-PGI), which uses proprietary integrases for high-precision DNA insertion, akin to text-pasting in a word processor. I-PGI has successfully inserted over 30kb of genetic code into various cell types and can be used for complex cell engineering.

This technology facilitates more controlled gene therapy, allowing natural promoters in the human genome to regulate gene expression. Tome initially focuses on therapies for monogenic liver diseases and autoimmune diseases.

The team, led by Dr. Rahul Kakkar, brings extensive experience in pharma, biotech, and gene therapy. Having raised $213M from prominent biotech investors like Andreessen Horowitz and ARCH, Tome's platform represents a significant advancement in genomic medicine with potential widespread therapeutic applications.

Highlights and risks

Valuation

Given the early stage of the company and limited information about its programs, we did not conduct a valuation analysis. The company has raised $213M to date and is thus likely valued between $300-500M.

Platform overview

Tome Bioscience's PGI platform builds on CRISPR technology and is characterized by several innovative components and functions. The platform is based on technology licensed from MIT, coined "Programmable Addition via Site-specific Targeting Elements" (PASTE).

The platform enables the integration of large pieces of DNA into any part of the genome through a combination of targeted CRISPR-Cas9 technology and serine integrase-mediated recombination. Here’s how the system works to achieve this:

• Targeting with CRISPR-Cas9 Nickase
• The system uses a modified version of the CRISPR-Cas9 system, known as a Cas9 nickase. Unlike the traditional Cas9 which creates double-strand breaks (DSBs), the nickase version makes single-strand nicks in the DNA. This avoids potential damage associated with double-strand breaks (DSBs).
• This nickase is guided to a specific genomic location by a guide RNA (gRNA). The gRNA can be designed to target virtually any location in the genome, providing the system with its universal targeting capability.
• Attachment Site-containing Guide RNA (atgRNA)
• The guide RNA in the PASTE system is engineered to contain attachment sites (like AttB sites). These sites act like "beacons" that are recognized by serine integrases.
• By incorporating these attachment sites into the gRNA, the system directs the serine integrase to the precise location in the genome where the nick has been made.
• Role of Serine Integrase
• Serine integrases are enzymes that naturally mediate the integration of DNA at specific sites in bacterial genomes.
• In the PASTE system, the serine integrase is repurposed to recognize the engineered attachment sites in the human genome (or the genome of the target organism).
• When the integrase encounters the attachment site in the gRNA and a corresponding attachment site (like AttP) in the payload DNA (the DNA to be integrated), it facilitates a recombination event, leading to the integration of the payload into the genome.
• Integration of the DNA Payload
• The DNA payload, which is the sequence intended for integration, is engineered to contain an AttP site, complementary to the AttB site in the gRNA.
• Upon recognition of these complementary sites, the integrase mediates a site-specific recombination event, integrating the DNA payload at the targeted location in the genome.
• Flexibility and Precision
• The flexibility of the CRISPR-Cas system to target virtually any genomic location, combined with the precision of serine integrase-mediated recombination, allows the PASTE system to integrate DNA sequences into any part of the genome.
• This method bypasses the need for DSBs and the associated risks of off-target effects and genomic instability, making it a safer and more precise tool for genome editing.

Advantages over other gene editing techniques

PASTE does not rely on creating DSBs, which minimizes unwanted DNA damage such as large deletions, translocations, and potential chromosomal rearrangements.

Unlike HDR (homology-directed repair) methods, PASTE is efficient in non-dividing cells, expanding its potential for therapeutic use in a wider range of cell types, including those predominantly non-dividing in the human body.

PASTE displays fewer off-target effects compared to other gene insertion methods like HITI (homology-independent targeted integration) due to the specificity provided by CRISPR-guided targeting and the use of circular DNA templates that limit random integration.

It has demonstrated high specificity and insertion purity, which are critical for therapeutic applications where accuracy and safety are paramount.

Prime editing allows for the introduction of small DNA edits (up to around 44 base pairs) without double-stranded DNA breaks. It is highly precise for small-scale edits but is not designed for integrating large DNA sequences. Base editors enable the conversion of one DNA base into another without making double-stranded DNA breaks, useful for correcting point mutations. However, they cannot be used to insert larger DNA sequences.

The combination of CRISPR-based targeting and serine integrase-mediated recombination in this system overcomes a limitation of both prime editing and base editing by enabling the insertion of much larger DNA sequences than what is possible with these methods. This capability is particularly significant for therapeutic applications where a functional gene or large DNA sequence needs to be integrated into the genome. It also potentially simplifies the integration process by using a single delivery mechanism to introduce both the integrase landing site and the DNA cargo.

Evaluating the technology

The company makes several assertions about the capability of the technology. Based on the publication provided by the company, here is the evidence provided for each assertion.

The technology can insert DNA sequences of any size into designated genomic locations.

Evidence within the paper supporting this assertion includes:

• The Results section mentions: "We demonstrate integration of sequences as large as ~36 kb at multiple genomic loci..."
• The Abstract states: "PASTE... allows for... integration of sequences as large as ~36 kb."
• Figure 1f-h and Extended Data Figure 2 show that they could achieve precise integration of templates as large as ~36,000 bp with PASTE.

It is important to be cautious in claiming that sequences of any size can be inserted, as the study demonstrates successful integration up to around 36 kb but does not empirically validate beyond that size. While the results are promising, they represent a subset of the potential sizes of DNA sequences.

Strengths of the Evidence:

• The experimental data consistently show that PASTE can integrate large sequences up to the demonstrated limit.
• The ability to integrate without relying on double-strand DNA breaks potentially reduces the risks associated with traditional genome editing, such as unintended mutations or genomic instability.
• Multiple loci and cell types, including non-dividing cells, were successfully targeted.

Weaknesses of the Evidence:

• "Any size" is a broad claim and technically has not been empirically proven beyond the ~36 kb size mentioned. It is unclear whether there is an actual upper limit, which could be influenced by various biophysical and cellular factors.
• The specificity and efficiency of integration for significantly larger sequences have not been evaluated.
• Biological systems' response to the insertion of very large DNA sequences, such as potential impacts on genome structure and function, has not been extensively discussed.
• Evidence is primarily based on in vitro experiments, with in vivo data limited to an animal model (humanized liver mice), which might not fully represent the response in humans.

In conclusion, while the evidence supports the assertion for sequences up to 36 kb, the claim that PASTE can insert sequences of any size into genomic locations requires cautious interpretation and further empirical support, especially for sequences larger than 36 kb. The technology is promising, but the biological complexities that come with genetic manipulation necessitate thorough understanding and careful claims.

The technology can insert DNA into any part of the genome

Evidence within the paper supporting the assertion

• The publication states that "We demonstrate integration of sequences as large as ~36 kb at multiple genomic loci..." (Results section and the Abstract), which implies targeting different parts of the genome.
• They have demonstrated successful genome insertion at various loci: "...we were able to achieve precise integration of templates as large as ~36,000 bp at nine different endogenous sites with 13 different cargos..." (Discussion section).
• The methods involve CRISPR-Cas9 technology, which is known for its ability to target specific genomic locations using RNA guides designed to match the DNA sequence of interest.

Strengths of the Evidence:

• PASTE has been demonstrated to be effective at different genomic loci, illustrating the technology's versatility in targeting.
• The combination of CRISPR-Cas9's targeting mechanism and serine integrase integration suggests a high degree of specificity could be achieved.

Weaknesses of the Evidence:

• While the technology has been demonstrated to work at multiple loci, the paper does not provide evidence that PASTE can target absolutely any genetic locus.
• Genomic regions vary in their accessibility and complexity; some loci might be difficult to edit due to structural aspects of the chromatin, the presence of repetitive elements, or regulatory implications.
• The integration efficiency could vary across different genomic contexts, which might limit the practical application of the technology at some genomic locations.
• In vivo validation of this broad assertion in complex organisms, including humans, is not yet described in the provided text.

In summary, while the technology has been shown to integrate DNA at various sites within the genome, the assertion that it can insert DNA into any part of the genome is overly broad and requires additional evidence. There remain technical, biological, and efficiency challenges associated with CRISPR-based genome editing that can affect targeting flexibility. It's essential to clarify these limitations when asserting the capabilities of PASTE to ensure accurate communication of the technology's potential.

Enables the insertion of DNA sequences without the formation of double-strand breaks (DSBs)

This assertion is consistent with various portions of the text:

• In the Abstract and throughout the Results section, it is highlighted that PASTE technology uses CRISPR-Cas9 nickase instead of a nuclease that creates double-strand DNA breaks, stating that "Programmable genome integration of large, diverse DNA cargo without DNA repair of exposed DNA double-strand breaks (DSBs) remains an unsolved challenge in genome editing. We present Programmable Addition via Site-specific Targeting Elements (PASTE), which uses a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase for targeted genomic recruitment and integration of desired payloads."
• The study also specifically mentions the integration of large sequences of up to ~36 kb without DSBs and outlines an alternative to relying on DSBs for genome editing, a process that often involves cellular DNA repair mechanisms like non-homologous end joining (NHEJ) or homology-directed repair (HDR).

Strengths of the Evidence:

• The publication presents data indicating successful integration of large DNA sequences without the use of DSBs, which is a significant technical advancement. Traditional CRISPR-Cas9 approaches typically rely on creating DSBs to induce repair mechanisms that can be co-opted for gene insertion.
• By employing a CRISPR-Cas9 nickase fused to a reverse transcriptase and serine integrase, PASTE illustrates a mechanism by which insertion can be achieved through a precise and ostensibly less mutagenic process.
• Avoidance of DSBs suggests potentially fewer off-target effects and unwanted genetic alterations that could arise from error-prone repair mechanisms like NHEJ.

Weaknesses of the Evidence:

• Although data support the assertion that PASTE can insert DNA sequences without DSBs, the provided text does not detail the efficiency and fidelity of this process across a broad range of genomic contexts and cell types, which might affect its practical utility.
• There might be specific genomic contexts or cell types where PASTE could be less effective or less practical due to reasons not related to DSBs (e.g., chromatin accessibility, cellular toxicity, or delivery challenges).
• The study could also benefit from further validation under a variety of conditions in vitro, in vivo, and across different organisms to ensure broad applicability of the findings.

Concluding, the evidence strongly supports the assertion that PASTE can insert DNA sequences without the need for double-strand breaks, which is a pivotal improvement over existing genome editing technologies that often rely on induction of DSBs. This evidence is a strength of the technology and represents an area where PASTE has a clear advantage. However, it is essential for future studies to explore the limits and contexts in which this technology functions most effectively without DSBs to fully harness its potential.

Unprecedented precision compared to existing tech including base editing and prime editing

Evidence within the paper supporting the assertion:

• The term "precision" in genome editing usually refers to the ability to insert DNA at the desired genomic location without unintended edits elsewhere in the genome. The Results section indicates that PASTE enables "integration of sequences as large as ~36,000 bp with ~10-20% integration efficiency at ACTB and LMNB1" and "can replace 130 bp and 385 bp of genomic sequence at a rate of 7-10%."
• The Abstract and the Results section report that PASTE exhibits "fewer detectable off-target events" compared to conventional methods like homology-independent targeted integration (HITI) and homology-directed repair (HDR).
• The paper compares PASTE to HITI and HDR methods. Figure 3 represents these comparisons, showing higher insertion purity and lower off-target effects.
• Specifically, the paper discusses that genome-wide off-target effects are significantly lower for PASTE than for HITI, supported by analyses presented in Figure 3 showing PASTE's 34.8% on-target editing with no detectable off-target activity in genome-wide assays, unlike HITI.

Strengths of the Evidence:

• The study provides empirical data demonstrating the PASTE system's ability to insert large sequences with high precision and a lower rate of unintended off-target effects compared to some other genome editing methods.
• The comparison to HITI and the references to fewer off-target events suggest that PASTE might offer improved precision over some existing technologies.

Weaknesses of the Evidence:

• The paper provides a limited direct comparison of PASTE to base editors and prime editors. While less focused on large insertions, base and prime editing techniques are known for their high precision in single base pair edits and small sequence alterations.
• There is not a comprehensive evaluation of the entire range of existing technologies, especially when base and prime editors are considered, which are designed for different types of edits rather than the direct insertions that PASTE performs.
• The efficiency and precision of PASTE in various genomic contexts and different cell or organism types remain to be thoroughly evaluated.

Based on the evidence within the publication, PASTE shows promising precision for large DNA insertions with potentially fewer off-target effects than some traditional genome editing technologies. However, the provided data do not sufficiently compare PASTE with the precision of base editing and prime editing for it to be declared unprecedentedly precise across the board. While PASTE may offer advantages for inserting large DNA sequences, base and prime editing's precision for their designed applications—smaller nucleotide changes without double-strand breaks—sets a high bar which PASTE does not directly challenge. In summary, while the assertion of high precision is supported concerning certain editing approaches, the term "unprecedented" should be used cautiously until further comparative data are available.

The technology can be multiplexed, placing multiple sequences simultaneously

• In the Results section, PASTE is described to have been engineered for "simultaneously introducing three genes at three separate loci" (Fig. 4g), suggesting the ability to multiplex.
• The paper discusses the identification of orthogonal integrase AttB/AttP sites that are specific to particular integrases. The Results section mentions that experiments demonstrated "multiplexed gene insertion with PASTE, enabling applications such as...fusion of three different endogenous genes with three different fluorescent cargos" (Discussion section).
• The methodology for achieving multiplexed insertion includes testing different central dinucleotide combinations for the AttB/AttP sites that affect integrase activity. The text in the Results section states: "Pairing tested GA, AG, AC, and CT dinucleotide atgRNAs with their corresponding AttP cargo... no detectable integration across mispaired combinations" (Fig. 4f).
• Multiplexed gene insertion was validated by "bulk genomic DNA harvesting and ddPCR, of all possible combinations" (Fig. 4g), as well as "multiple applications for gene insertion and tagging" (Discussion section).

Strengths of the Evidence:

• Specific experiments directly demonstrate the capability for multiplexed genome editing with PASTE by inserting different sequences into distinct loci.
• The development and validation of orthogonal integrase recognition sites enable specific integrases to act independently without cross-reactivity, a key for multiplexing.
• The ddPCR and NGS data presented provide robust, quantitative methods for confirming multiplexed insertions.

Weaknesses of the Evidence:

• While the examples in the text demonstrate that multiple genes can be inserted concurrently, they may not cover the full potential or limits of multiplexing. The exact number of insertions that can be multiplexed simultaneously while maintaining high efficiency and specificity is not established.
• Multiplexing efficiency might vary with different cargo sizes, cell types, genomic targets, or overall experimental conditions, which are not fully explored in the provided text.
• Potential challenges associated with the delivery of multiple components needed for PASTE multiplexing (e.g., multiple guides, templates, and integrases) into the same cell are not extensively discussed.

In conclusion, the publication provides solid evidence that PASTE can be multiplexed to place multiple sequences simultaneously at distinct genomic locations, showcasing one of its potential strengths. Multiplexed editing demonstrated in the paper represents an advance in gene insertion technology. However, more data would be beneficial to define the range and limitations of multiplexing capability more precisely and to ensure robust performance across varying conditions.

The technology can edit dividing and non-dividing cells

• In the Results section, it is stated that PASTE "functions in non-dividing and primary cells," indicating that the technology is not solely reliant on cellular processes associated with cell division.
• The Abstract mentions that PASTE "has activity in non-dividing cells," again supporting this assertion.
• Non-dividing HEK293FT cells were successfully edited using PASTE, as shown in Extended Data Figure 9a-d, where it is reported that the technology maintained gene integration activity of greater than 20%.
• The Results section also states that PASTE achieved "around 5% efficiency in primary human T cells" and "primary human hepatocytes."

Strengths of the Evidence:

• There are specific experiments presented in the paper that show successful PASTE-mediated DNA insertion in non-dividing cells, supporting the notion that the technology does not rely on cellular DNA repair mechanisms that are typically more active in dividing cells.
• The ability to edit non-dividing cells is a significant advantage and is particularly relevant for potential therapeutic applications involving post-mitotic tissues such as neurons and muscle cells.
• The inclusion of primary human hepatocytes in the validation experiments adds to the clinical relevance of the technology, as the liver is a key target for many gene therapy applications.

Weaknesses of the Evidence:

• The paper reports efficiencies for non-dividing cells that are lower than those observed for dividing cells, which is particularly evident when looking at the reported ~5% efficiency in primary human hepatocytes. This suggests there may be efficiency limitations when working with non-dividing cell types.
• Efficiency in non-dividing cells appears to vary based on cell type, as indicated by the data showing different integration rates in different non-dividing cells.
• The paper provides a relatively small dataset for primary human T cells and hepatocytes, and a broader range of non-dividing cell types should be tested to fully confirm the technology's versatility in this context.
• The in vivo context and therapeutic settings, which are important for the application of this technology, are not discussed extensively in relation to non-dividing cells. Extended data Figure 10 discusses some in vivo testing in liver-humanized mice, but further validation in a broader set of tissues and in vivo models would strengthen the evidence.

In summary, the evidence provided clearly demonstrates that PASTE can be used to edit both dividing and non-dividing cells, supporting the assertion. This capability broadens the potential applications of this technology, especially in the context of tissue and cell types that do not proliferate regularly. However, further research to optimize efficiency and validate the technology's efficacy across various non-dividing cell types and in vivo settings would be beneficial.

The technology has fewer off-target edits than other approaches

Evidence within the paper supporting the assertion:

• The Results section and Figure 3 report that PASTE has less off-target activity and generates fewer indels compared to HITI: "We also used this genome-wide pipeline to analyze HITI off-targets using the same ACTB guide and HITI EGFP insertion template and found substantial off-target activity across the genome..." In contrast, for PASTE, “...all reads aligning to the on-target ACTB site, confirming no off-target genomic insertions”.
• The Results section describes that PASTE has better gene insertion efficiencies and purity compared to HITI and HDR at the ACTB locus, with significantly fewer indels: "PASTE had better gene insertion efficiencies than HITI... significantly fewer indels generated with PASTEv3 than HITI in both HEK293FT and HepG2 cells...".

Evidence not addressed within the paper:

• The paper does not directly compare PASTE off-target edits to those observed with base editing and prime editing technologies. These methods have different modes of action and have been optimized for high precision edits without inducing DSBs. Specifically, base editing directly converts one base pair to another without creating a break, while prime editing makes single-base substitutions or small insertions/deletions using a nick and reverse transcription rather than a DSB.
• Base editing and prime editing have been characterized in independent studies to have low off-target effects. The assertion that PASTE has fewer off-target effects than these technologies would require head-to-head comparisons, showing quantitative evidence of off-target edits using a method like deep sequencing or unbiased genome-wide off-target analysis.

Strengths of the Evidence:

• PASTE’s off-target performance relative to HITI or HDR, techniques that rely on DSBs, is clearly demonstrated to be superior within the context of the experiments performed.
• The avoidance of DSBs in the editing mechanism of PASTE itself logically implies a reduced likelihood of inducing the kind of genomic instability associated with conventional nuclease-driven editing.

Weaknesses of the Evidence:

• Lack of direct comparison to base or prime editing leaves the assertion unfounded within the context of those specific technologies.
• The phrase "other approaches" is broad and may sweepingly encompass technologies that are not strictly comparable due to different applications and mechanisms.
• Off-target effects can be location-specific, dependent on various factors including the guide RNA used, so broad claims about off-target effects need extensive validation across different genomic contexts.

In conclusion, while the paper demonstrates that PASTE has a specificity profile that may be more favorable than methods using DSBs like HITI, the assertion that it has fewer off-target edits compared to base editing or prime editing is not directly supported by the provided evidence. Comparative, comprehensive analyses would be required to fairly assess the precision of PASTE relative to these other highly precise genome editing technologies.

Addressing the limitations of gene editing

Gene editing technologies, including traditional CRISPR-based approaches and newer methods like base editing and prime editing, have transformed biomedical research and hold immense potential for treating genetic disorders. However, they also come with several limitations and challenges:

• Off-Target Effects: One of the major concerns with CRISPR and other gene editing technologies is off-target editing. This occurs when the editing machinery modifies DNA sequences other than the intended target, potentially leading to unintended genetic alterations, which can cause harmful effects in cells or organisms.
• Efficiency and Precision of Editing: While technologies like CRISPR/Cas9 are relatively efficient, the precision of editing can vary. Achieving high-efficiency editing with minimal off-target effects remains a challenge. Techniques like base editing and prime editing aim to improve precision but are not foolproof.
• Delivery into Target Cells and Tissues: Efficiently delivering gene editing tools into specific types of cells and tissues in a living organism is a significant challenge. Different delivery methods (like viral vectors, nanoparticles, and physical methods) have their limitations in terms of efficiency, safety, and tissue specificity.
• NHEJ vs. HDR: Non-homologous end joining (NHEJ) and homology-directed repair (HDR) are two pathways that cells use to repair double-strand breaks induced by CRISPR. NHEJ is error-prone and can lead to insertions or deletions (indels) at the target site. HDR, while more precise, has a lower efficiency and is more challenging to control.
• Immune Response: The introduction of foreign genetic material and proteins (like Cas9) into the body can trigger an immune response. This can reduce the effectiveness of the treatment and can also lead to adverse effects in patients.
• Ethical and Regulatory Concerns: The use of gene editing in human embryos and germline cells raises ethical concerns, including issues around consent, potential for misuse, and long-term effects on the human gene pool. Regulatory frameworks for gene editing therapies are still evolving, with varying standards across different countries.
• Genetic Complexity and Mosaicism: Many diseases are caused by complex genetic factors and not just single gene mutations. Addressing these complex genetic diseases with gene editing poses significant challenges. Additionally, in some cases, gene editing can lead to mosaicism, where some cells are edited, and others are not, leading to variable outcomes.
• Long-term Effects and Stability: Understanding the long-term effects of gene editing in living organisms, including potential unintended consequences, is crucial. The stability of the edits over time and across cell divisions is also a concern, especially for treatments aimed at long-lasting or permanent correction of genetic disorders.
• Accessibility and Cost: The development, production, and application of gene editing therapies are currently expensive and complex, limiting their accessibility. The cost and expertise required to develop and administer these therapies make them less accessible, especially in low-resource settings.
• Base Editing and Prime Editing Specific Challenges: While these newer techniques offer improved specificity and reduced off-target effects, they also have their own limitations. Base editors have a limited editing window and can only make specific types of nucleotide changes. Prime editing is more versatile but is also more complex and less efficient compared to traditional CRISPR/Cas9.

Potential of PGI to address limitations

Below is discussion of the evidence supporting PGI's ability to overcome the limitations associated with gene editing. Some of these have already been discussed above, so I'll focus on limitations that haven't already been covered.

Drawing on evidence provided in the paper describing Tome's technology, I will look for evidence within the paper regarding PASTE's potential to overcome the limitation of off-target effects, common to CRISPR and other gene editing approaches:

Delivery into Target Cells and Tissues

Evidence within the paper:

The technology developed by Tome Biosciences, as detailed in the paper, addresses the challenge of delivering gene editing tools into target cells and tissues with the following evidence:

• PASTE was demonstrated to function in both dividing and non-dividing cells, including primary human hepatocytes and T cells.

• The paper mentions the delivery of PASTE components using adenoviral (AdV) vectors, which are noted to be capable of packaging large cargos, thus accommodating the size requirements of PASTE components for efficient delivery.

• The use of programmable genome insertion without DNA repair pathways suggests that PASTE could be active across a range of cell types, potentially translating to various tissues in vivo.

• In the paper, the researchers showed successful in vivo programmable gene insertion in the liver using PASTE components delivered via adenoviral vectors in mouse models.

• There is mention of compatibility with different viral delivery methods, including adeno-associated virus (AAV) and adenovirus (AdV), to transport DNA integration templates, broadening the delivery options.

Strengths of the evidence:

• Efficacy in a wide range of cells, including non-dividing primary human hepatocytes and T cells, indicates a broad potential for reaching different tissues.

• Adenoviral delivery is a well-established method for in vivo gene therapy and the efficient transduction achieved is a strong point of evidence.

• The successful demonstration of potential in vivo gene insertion in the liver of a mouse model raises confidence in the versatility and translatability of the PASTE delivery system.

Weaknesses of the evidence:

• While the data indicate that PASTE components can be delivered via viral vectors in vitro and in mouse models, the safety, efficiency, and potential immunogenicity of such vectors in humans are not addressed in the paper.

• Data on tissue-specific delivery in vivo are limited to the liver, and a broader range of tissues needs to be assessed to support the claim of wide applicability.

• AAV vectors, although mentioned, are known for their limited packaging capacity, which could pose a constraint for delivering larger gene editing tools.

• The long-term expression and potential integration of viral vector genomes into the host genome, which is a concern for AdVs, are not discussed.

Potential of the technology to overcome the limitation:

PASTE shows promise in overcoming the challenge of delivering gene editing tools into target cells and tissues, primarily due to its adaptability with viral vectors known for efficient delivery. The successful liver-targeted gene insertion in vivo signals potential applicability for therapeutic gene editing. However, additional research into alternate delivery systems, like non-viral vectors or physical methods (e.g., electroporation), could further augment delivery options and address limitations related to safety and specificity.

Immune Response

Evidence within the paper:

The paper mentions the consideration of immune responses to the components of the PASTE system, as outlined below:

• The use of adenoviral (AdV) vectors and adeno-associated virus (AAV) vectors for delivery: While AdVs are known to elicit an immune response, AAVs are generally characterized by lower immunogenicity. The publication discusses the compatibility of PASTE with these vectors, suggesting that AAVs could be a less immunogenic option for delivering PASTE components.

• Transcriptome analysis of cellular responses to PASTE: The paper states that transcriptome-wide analysis was conducted to assess off-target effects of expressing PASTE components in HEK293FT cells, with very few changes observed in gene expression, potentially indicating a limited cellular stress response.

• PASTE in vivo testing: The in vivo application of PASTE in liver-humanized mouse models (specifically the FRG KO mouse model) is mentioned, which can provide preliminary data related to immune responses in a living organism, although the focus is on integration efficiencies and not the immune reaction per se.

Strengths of the evidence:

• The consideration of different viral vectors for PASTE component delivery expands the range of options, potentially allowing for tailoring the delivery method to reduce immunogenicity.

• The observation of minimal transcriptome changes when expressing PASTE components suggests a reduced stress or immune response to the system within the context of the tested cell type.

Weaknesses of the evidence:

• While AAVs generally have a lower immunogenic profile, the response can be different in humans, and the long-term immunogenicity of PASTE components in clinical settings remains unknown.

• Transcriptome analysis, although providing initial insights into cellular responses, does not directly address adaptive immune responses that could be triggered by the introduction of PASTE components into humans.

• Data from in vivo mouse model experiments, while essential, may not fully represent the immune responses in humans, and further testing in more relevant models would be necessary.

• There is no detailed discussion about whether PASTE components themselves may be immunogenic or if they could alter cell surface markers and thereby impact immune recognition.

Potential of the technology to overcome the limitation:

The PASTE technology has the potential to mitigate immune responses, especially if compatible with AAV vectors known for low immunogenicity, and assuming that the PASTE components themselves do not provoke significant immune reactions. However, this potential is primarily speculative at this stage and requires in-depth investigation.

Genetic Complexity and Mosaicism

Evidence within the paper:

The PASTE technology potentially enables the integration of large sequences at targeted genomic locations, which might be beneficial in addressing genetic complexity by allowing the insertion or replacement of multigene segments. The key aspects from the paper concerning this are:

• PASTE's ability to integrate sequences as large as ~36 kb at multiple genomic loci across cell types suggests it can be utilized to deliver complex genetic cassettes required to address multigenic disorders.

• The precision of PASTE, facilitated by fusion with serine integrases, can potentially prevent mosaic outcomes, as it promotes site-specific integration rather than random insertion.

• PASTE was shown to perform multiplexed gene integration by introducing three genes at three separate loci, indicating its capacity for complex genetic manipulations.

• The successful use of PASTE in non-dividing primary human hepatocytes indicates that it might overcome issues related to cell division, which can contribute to mosaicism in gene-edited populations.

Strengths of the evidence:

• The ability to deliver large genetic sequences implies that PASTE could be utilized for complex genetic engineering scenarios, including multigene disorders.

• The demonstrated multiplexing capability is a significant advantage when tackling diseases caused by mutations in multiple genes or those that require the introduction of several genetic elements.

Weaknesses of the evidence:

• While the paper describes the introduction of large and multiplex edits, the functional outcomes of these edits and their potential to address complex genetic diseases are not characterized.

• The extent to which PASTE avoids mosaicism has not been thoroughly demonstrated. While site-specific insertions might increase homogeneity, comprehensive analysis of editing outcomes at the cellular population level was not presented.

• There is no discussion on the regulation of the inserted genes, which is crucial for diseases with complex regulatory networks that cannot be addressed by gene insertion alone.

Potential of the technology to overcome the limitation:

PASTE's potential to accommodate larger genetic payloads and enable multiplex editing suggests it could be used to address genetic complexity in diseases. Moreover, its site-specific nature might reduce mosaicism. However, the actual performance of PASTE in tackling complex genetic diseases and the prevention of mosaicism need to be proven in disease-relevant model systems, particularly for polygenic conditions.

Long-term Effects and Stability

Evidence within the paper:

The paper provides some insights into the long-term effects and stability of the gene editing technology known as PASTE:

• PASTE does not rely on DSBs or HDR, suggesting that the edits introduced should, in theory, be more stable due to the avoidance of error-prone repair pathways.

• Multiplexed gene integration capability is shown, which—if stable—could demonstrate PASTE’s potential for long-lasting genetic modification.

• For evaluating the stability of the integration, the authors present data where they test the targeting of genes with larger cargos (up to ~36 kb) and data showing in vivo experiments in liver-humanized mice.

• PASTE is said to have fewer detectable off-target events, which could point to less genomic instability overall post-editing.

Strengths of the evidence:

• The technology's ability to integrate large DNA fragments without relying on error-prone DNA repair mechanisms enhances the prospect of achieving stable genetic modifications.

• The paper provides initial in vivo validation in liver-humanized mice, which indicates the potential for long-term stability of the edits.

Weaknesses of the evidence:

• The publication primarily focuses on the efficiency and precision of the PASTE technology, with less emphasis on the long-term implications of genomic alterations.

• There is limited information on the consistency of edit stability through multiple rounds of cell division, particularly in tissues with high cellular turnover.

• Findings from the liver-humanized mice cannot be directly extrapolated to humans or to other tissues and cell types.

• The evidence does not explicitly address the persistence of edits in a clinical context, nor does it mention the impact on epigenetics or the possible genotoxic effects of the integrated DNA sequences over an extended period.

Potential of the technology to overcome the limitation:

PASTE potentially provides a stable alternative for gene editing, especially for permanent correction of genetic disorders. Its design minimizes reliance on cellular DNA repair pathways, which can increase the fidelity and stability of edits. However, the long-term effects, particularly regarding safety, stability across cell divisions, and in different tissues, require further validation.

Base Editing and Prime Editing Specific Challenges

Evidence within the paper:

The PASTE technology described in the paper addresses some of the limitations associated with base and prime editing, as detailed below:

• Large Payload Integration: PASTE demonstrates its capability to integrate large sequences (~36 kb) with efficiencies up to ~50-60% in cell lines, which surpasses the current capabilities of base and prime editors that are restricted to small nucleotide changes and short insertions or deletions.

• Editing Versatility: The paper outlines PASTE’s ability to efficiently target multiple loci for integration, suggesting a potential advantage over base editing’s limited editing window. PASTE's use of integrases with various recognized attachment sites also presents an opportunity to target distinct loci within the genome.

• Multiplexed Editing: The paper shows PASTE performing multiplexed integration by simultaneously introducing three genes at three separate loci, which is beyond the current scope of base or prime editing that typically targets single loci.

• Strengths of the evidence:

  • The multi-kb insertions capability of PASTE is a significant advance over base and prime editing's limits in terms of the size and complexity of genetic material that can be added to the genome.

  • PASTE’s versatility in targeting and efficiency points towards substantial improvements over prime editing in these respects.

  Weaknesses of the evidence:

  • The paper doesn’t compare PASTE directly with base editors or prime editors, so there’s no direct evidence that PASTE is more effective in scenarios where base or prime editing would traditionally be used (e.g., single or few base pair edits).

  • While prime editing is known to be less efficient, PASTE does not elucidate its efficiency rates in comparison to prime editing at a granular level.

  • The complexity of PASTE’s delivery is acknowledged, involving multiple components (Cas9 nickase, reverse transcriptase, integrase). This added complexity could be comparable to prime editing’s more intricate system, but the paper does not discuss this aspect in detail.

  Potential of the technology to overcome the limitation:

  PASTE appears to have the capacity to overcome some of the challenges specific to base and prime editing, particularly those related to the size of the editable genetic material and the ability to perform multiplexed edits. Its approach, which circumvents DSBs, also stands to potentially offer a more precise and less-damaging method of genome modification.

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  Market overview

  
  

  Tome says its initial therapeutic focus is on gene therapy for monogenic liver disease and cell therapy for autoimmune disease.

  
  

  Liver disease

  
  

  The rationale for using gene therapy and gene editing in treating liver diseases is based on several key factors. The liver has a remarkable ability to regenerate, making it a resilient target for gene therapy. Modified cells can proliferate, enhancing the effectiveness of the treatment. The liver plays a crucial role in metabolism, including protein synthesis and detoxification. Correcting genetic defects in liver cells can restore normal function and prevent systemic complications. The liver is readily accessible for gene delivery, especially through the bloodstream. This makes it easier to deliver genetic material or gene-editing tools directly to liver cells. The liver has a unique immune-tolerant environment, which can reduce the likelihood of immune reactions against the introduced genetic material, making gene therapy safer and more effective.

  TOME Biosciences' Programmable Genomic Integration (PGI) technology, particularly advantageous for treating monogenic liver diseases, could excel in addressing conditions that are challenging for existing gene editing technologies like CRISPR/Cas9, base editing, and prime editing. The key advantages of PGI, such as the ability to insert large DNA sequences with high precision and avoid double-strand DNA breaks, position it uniquely for certain diseases. Here are a few monogenic liver diseases where Tome's technology might have a particular advantage:

  • Alpha-1 Antitrypsin Deficiency (AATD): AATD is caused by mutations in the SERPINA1 gene, leading to liver and lung disease. Traditional gene editing approaches may struggle with precise insertion of the large SERPINA1 gene or correcting the diverse mutations seen in AATD. PGI's ability to insert large DNA sequences precisely could enable the addition of a functional SERPINA1 gene, potentially offering a curative approach.
  • Wilson's Disease: This is caused by mutations in the ATP7B gene, leading to copper accumulation in the liver and other tissues. The ATP7B gene is large, and patients have a wide range of mutations, making precise gene correction challenging. PGI could insert a functional ATP7B gene, circumventing the need for specific mutation targeting.
  • Hereditary Hemochromatosis: Caused by mutations in the HFE gene, leading to iron overload. While some mutations are common (like C282Y), others are rare and diverse. PGI could introduce a functional HFE gene, providing a treatment that doesn't rely on correcting specific mutations.
  • Citrullinemia Type I: Caused by mutations in the ASS1 gene, leading to ammonia accumulation. Base and prime editing might not effectively address all the mutations in the ASS1 gene, but PGI could add a functional ASS1 gene to restore normal function.
  • Progressive Familial Intrahepatic Cholestasis (PFIC): This group of disorders is caused by mutations in several genes (like ATP8B1, ABCB11, and ABCB4) involved in bile formation. Given the complexity of these genes and the diversity of mutations, inserting functional copies of these genes using PGI could be a more effective strategy than trying to correct specific mutations.
  • Glycogen Storage Diseases: Such as Type IV (caused by mutations in the GBE1 gene). These diseases are characterized by the accumulation of abnormal glycogen in the liver. Base and prime editing might not be feasible for all GBE1 mutations, but PGI could potentially introduce a functional copy of the gene.
  
  
  

  Disease Name	Description	Associated Gene(s)	Standard of Care	Prognosis	Unmet Need
  Alpha-1 Antitrypsin Deficiency (AATD)	A genetic disorder causing liver and lung disease due to alpha-1 antitrypsin protein deficiency.	SERPINA1	Augmentation therapy, bronchodilators, and pulmonary rehabilitation for lung issues; liver transplantation in severe cases.	Variable; lung disease can be severe; liver disease may lead to cirrhosis; not all patients develop liver disease.	Effective treatments for lung and liver aspects; gene therapy to correct underlying genetic defect.
  Wilson's Disease	Copper accumulation in liver, brain, and other organs, leading to liver disease and neurological symptoms.	ATP7B	Chelating agents to remove excess copper, zinc therapy, and in severe cases, liver transplantation.	Good with early treatment; poor if untreated leading to liver failure or severe neurological damage.	Therapies with fewer side effects; treatments addressing neurological symptoms more effectively; earlier diagnosis.
  Hereditary Hemochromatosis	Excessive iron absorption causing liver damage, heart problems, and diabetes.	HFE, HJV, HAMP, TFR2	Phlebotomy, iron chelation, and management of complications like liver disease and diabetes.	Generally good with early diagnosis and treatment; can be poor if complications like cirrhosis develop.	Non-invasive diagnostic methods; broader screening; therapies to prevent organ damage.
  Citrullinemia Type I	Ammonia accumulation due to urea cycle deficiency.	ASS1	Low-protein diet, medications to remove ammonia, liver transplantation in severe cases.	Variable; can be severe with neurological damage if untreated; better with early intervention.	Effective long-term treatments; gene therapies to address the underlying cause.
  Progressive Familial Intrahepatic Cholestasis (PFIC)	Rare liver disorders disrupting bile flow in children, leading to liver damage.	ATP8B1, ABCB11, ABCB4	Medications to reduce itching and cholestasis, vitamin supplementation, liver transplantation in severe cases.	Variable; can progress to liver failure requiring transplantation.	Treatments to prevent progression; better management of symptoms.
  Glycogen Storage Diseases	Disorders affecting sugar use and glycogen storage, causing liver enlargement and metabolic problems.	Various, including G6PC, SLC37A4, AGL, GBE1, GAA, PYGL, PFKM	Dietary management, supplements, medications to control symptoms, liver transplantation for specific types.	Depends on type; can range from relatively mild to life-threatening.	Therapies to correct the underlying metabolic defects; treatments to improve quality of life; gene therapies.

  
  

  B cell autoimmune disease

  
  

  B cells play a crucial role in the immune system, including the production of antibodies and presentation of antigens. In autoimmune diseases, these cells can become dysregulated, attacking the body's own tissues. Cell therapy can be designed to selectively target and modulate these aberrant B cell functions, restoring normal immune system balance.

  Traditional treatments for autoimmune diseases often involve broad-spectrum immunosuppressants, which can have significant side effects and increase infection risk. Cell therapies could offer a more targeted approach, potentially reducing the need for long-term systemic immunosuppression.

  Cell therapies, especially those that are genetically edited, can be engineered to target specific B cell populations or pathways involved in the autoimmune process. This specificity has the potential to increase the efficacy of the treatment compared to broader immunosuppressive therapies.

  However, there are several limitations and challenges with the current approaches to cell therapy for B cell autoimmune diseases:

  • Complexity of Autoimmune Diseases: Autoimmune diseases are often complex and can vary significantly between individuals. Developing cell therapies that effectively target the diverse mechanisms involved in these diseases is challenging.
  • Delivery and Persistence: Ensuring that the modified cells are efficiently delivered to the appropriate sites in the body and persist long enough to have a therapeutic effect is a significant challenge.
  • Risk of Unintended Immune Reactions: Anytime cells are manipulated and reintroduced into the body, there is a risk of unintended immune reactions, including the development of new autoimmune responses or rejection of the modified cells.
  • Off-Target Effects: Genetic editing techniques, while precise, can have off-target effects that might lead to unexpected consequences, including oncogenesis or disruption of normal immune functions.
  • Regulatory and Manufacturing Challenges: Developing cell therapies involves complex manufacturing processes, and these therapies must undergo rigorous testing and regulatory approval, which can be time-consuming and costly.
  • Accessibility and Cost: Cell therapies are often expensive to produce and may not be accessible to all patients who need them due to cost or healthcare infrastructure limitations.

  Tome Biosciences' Programmable Genomic Integration (PGI) technology could potentially overcome several limitations of current approaches in treating B cell autoimmune diseases:

  • Precision and Specificity: PGI technology, leveraging the site-specificity of CRISPR/Cas9 combined with proprietary integrases, enables the precise insertion of any DNA sequence into specific genomic locations. This precision is crucial in autoimmune diseases where the dysregulation of specific genes or pathways in B cells needs to be addressed without affecting other functions.
  • Large DNA Sequence Insertion: Traditional gene therapies often face challenges with inserting large DNA sequences. Tome's PGI can insert sequences of substantial length (more than 30kb), which could be crucial in introducing entire genes or regulatory elements necessary for reprogramming B cells in autoimmune conditions.
  • Reduced Off-Target Effects and Safety: Unlike traditional CRISPR/Cas9, which relies on double-strand DNA breaks (DSBs) and can lead to unintended mutations, Tome's approach minimizes unwanted DNA damage. This safety profile is particularly important in treating autoimmune diseases, where off-target effects can exacerbate the condition.
  • Efficiency in Non-Dividing Cells: PGI's efficiency in both dividing and non-dividing cells expands its applicability. Many cell therapy approaches struggle with targeting non-dividing cells, which is a significant limitation in autoimmune diseases where the affected cells might not be actively dividing.
  • Potential for Curative Treatment: By directly correcting the genetic basis of the autoimmune response in B cells, PGI offers a potential curative approach rather than just managing symptoms. This is a significant advancement over current treatments that often focus on systemic immunosuppression or symptomatic relief.
  • Flexibility and Customization: PGI allows for the customization of therapies for individual patients. Autoimmune diseases often exhibit significant heterogeneity among patients; this technology's flexibility could lead to more personalized and effective treatments.
  • Overcoming Delivery Challenges: The ability to reprogram cells ex vivo and then reintroduce them into the patient can potentially overcome some of the delivery challenges associated with in vivo gene therapy, ensuring that the modified cells are effectively targeted to the site of action.

  In conclusion, Tome's PGI technology addresses key challenges in treating B cell autoimmune diseases, including precision, safety, efficiency, and the potential for curative therapies. It represents a significant advancement in the field of cell and gene therapy, particularly for complex conditions like autoimmune diseases.

  • Rheumatoid Arthritis (RA): An autoimmune disorder that mistakenly attacks the joints. PGI could modify B cells to reduce autoimmune activity.
  • Systemic Lupus Erythematosus (SLE): Characterized by the immune system attacking its own tissues, affecting various organs. Targeted cell therapies could regulate abnormal B cell responses.
  • Multiple Sclerosis (MS): Although primarily T-cell mediated, B cells also play a role in MS. Modifying B cells could alter their contribution to the disease process.
  • Type 1 Diabetes (T1D): An autoimmune disease where the immune system attacks insulin-producing pancreatic cells. PGI technology could engineer B cells to regulate or suppress this response.
  • Myasthenia Gravis: A chronic autoimmune neuromuscular disease. Modifying the B cells involved could be a therapeutic strategy.
  • Sjögren's Syndrome: Attacks glands producing saliva and tears. Cell therapy could target the aberrant B cells responsible for autoantibody production.
  • Hashimoto's Thyroiditis: The immune system attacks the thyroid gland. Therapies could modulate the B cell response against thyroid antigens.
  • Graves' Disease: Leads to overactivity of the thyroid gland. B cell targeted therapies could modulate the immune response.
  • Pemphigus Vulgaris: A rare autoimmune disorder affecting skin and mucous membranes. B cell-targeted therapies could reduce autoantibody production.

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