Recombinant Pig V (D)J recombination-activating protein 1 (RAG1), partial

What is the structural organization of porcine RAG1 and how does it differ from human RAG1?

Porcine RAG1, like its human counterpart, consists of core and non-core regions. The core region (approximately aa 384-1040) maintains catalytic activity essential for V(D)J recombination, while the non-core region (particularly N-terminal aa 1-383) serves regulatory functions . The N-terminal domain (NTD) contains multiple zinc-binding motifs, including a Really Interesting New Gene (RING) domain (aa 287-351) with ubiquitin ligase activity . Within the non-core region, a specific segment (aa 1-215) facilitates interaction with DCAF1, leading to RAG1 degradation through CRL4-dependent mechanisms . Sequence alignment studies indicate high conservation of functional domains between porcine and human RAG1, especially in catalytic residues like D600, D710, and E964 that are critical for nuclease activity .

What phenotypes are observed in pigs with complete versus partial RAG1 deficiency?

Pigs with complete RAG1 deficiency (biallelic knockout) exhibit severe combined immunodeficiency (SCID) with characteristic T-B-SCID phenotype. These animals demonstrate hypoplasia of immune organs (thymus, spleen, and lymph nodes), complete absence of mature B and T lymphocytes, and failure to perform V(D)J recombination . In contrast, partial RAG1 deficiency in pigs, mimicking human hypomorphic mutations, shows a more variable phenotype with reduced but not absent lymphocyte populations, dysregulated peripheral lymphocyte development, and potential for autoimmunity . The manifestation of partial deficiency depends on the specific mutation and the remaining RAG1 activity, with some animals showing Omenn syndrome-like features including oligoclonal T-cell expansion and elevated IgE levels .

How do RAG1 and RAG2 function together in V(D)J recombination in the porcine immune system?

RAG1 and RAG2 form a heterotetramer that functions as an endonuclease in V(D)J recombination. In pigs, as in other vertebrates, this complex binds to recombination signal sequences (RSSs) flanking gene segments and catalyzes DNA cleavage through a mechanism requiring synapsis of two RSSs . RAG1 provides the catalytic core while RAG2 enhances specificity and efficiency. The complex generates double-strand breaks at RSS-coding sequence junctions, which are subsequently processed and joined by non-homologous end joining repair factors to create diverse immunoglobulin and T-cell receptor genes . In porcine cells, mutation of either RAG1 or RAG2 results in failed V(D)J recombination, demonstrating their interdependent relationship, with neither protein capable of completing recombination in the absence of its partner .

What are the optimal methods for generating RAG1 knockout pigs?

Current optimal methods for generating RAG1 knockout pigs involve CRISPR/Cas9-mediated gene editing combined with somatic cell nuclear transfer (SCNT). Based on recent successful studies, the following methodology yields high efficiency:

• Design guide RNAs (gRNAs) targeting conserved regions of RAG1 exon 2, particularly the region encoding the catalytic domain .

• Generate cell lines with targeted mutations through transfection of porcine fetal fibroblasts with Cas9 and gRNA expression vectors, followed by selection and screening .

• For homozygous knockout production, typical protocols include:

  • Transfection with CRISPR/Cas9 components

  • Single-cell colony isolation and expansion

  • Genotyping via PCR and sequencing to confirm biallelic modifications

  • Use of validated knockout cells as nuclear donors for SCNT

The efficiency of this approach has been demonstrated in multiple studies, with reports of successful generation of RAG1-deficient piglets at rates of 0.72-10% (calculated as knockout piglets per transferred embryos) . When designing CRISPR targets, researchers should focus on the 5' region of exon 2 to ensure complete disruption of the RAG1 open reading frame .

How can researchers verify RAG1 knockout efficiency in porcine models?

Verification of RAG1 knockout requires a multi-parameter assessment approach combining genomic, protein, and functional analyses:

• Genomic verification:

  • PCR amplification of the targeted region followed by sequencing to confirm mutations

  • Primers designed to detect both wild-type and mutant alleles (e.g., P1: 5′-TAGTACTTGGACTGCCTGGC-3′ and P2: 5′-GGCATGCATCGATAGATCTCG-3′)

• Protein expression analysis:

  • Western blotting of thymic or lymphoid tissue lysates to confirm absence of RAG1 protein

  • Immunohistochemistry to assess RAG1 expression in lymphoid tissues

• Functional verification:

  • Flow cytometry to quantify B and T lymphocyte populations (CD3+ T cells and IgM+ B cells should be dramatically reduced)

  • V(D)J recombination assay using PCR to detect recombination events at immunoglobulin and T-cell receptor loci

  • Histological examination of primary and secondary lymphoid organs for hypoplasia and absence of mature lymphocytes

Complete verification should include all three levels of assessment to conclusively demonstrate functional RAG1 deficiency.

What approaches are available for gene correction of RAG1 mutations in porcine models?

Several approaches have been developed for correcting RAG1 mutations in porcine models, with homology-directed repair (HDR)-mediated gene editing showing the most promise:

• Lentiviral vector-mediated gene therapy:

  • MND-c.o.RAG1 lentiviral vectors have shown partial restoration of immune function in mouse models with hypomorphic RAG1 mutations

  • This approach maintains physiological regulation of RAG1 expression

• CRISPR/Cas9-mediated HDR:

  • Exonic insertion strategies have been more successful than intronic approaches

  • In-frame insertion into exon 2 has been demonstrated to drive physiologic human RAG1 expression and activity

  • This technique allows disruption of dominant-negative effects of hypomorphic alleles

• Optimization parameters for successful correction:

  • Correction efficiency is enhanced by increasing HDR efficiency through cell cycle synchronization

  • Off-target effects should be minimized by careful gRNA design and use of high-fidelity Cas9 variants

  • Knock-in of RAG1 into safe harbor sites provides an alternative to direct correction

The choice of approach depends on the specific mutation and research goals, with HDR-mediated correction being most suitable for precise modification of the endogenous locus .

How does the non-core region of RAG1 regulate off-target V(D)J recombination?

The non-core region of RAG1 plays a crucial role in preventing off-target V(D)J recombination through multiple mechanisms:

• Chromatin binding and targeting:

  • The N-terminal domain (NTD) facilitates proper chromatin binding and genomic targeting of the RAG complex

  • Deletion of RAG1's non-core region results in more indiscriminate binding to the genome

• Regulation of recombination pathways:

  • The first 215 residues of RAG1 affect the balance between short-range and long-range recombination

  • R1Δ215 mice exhibit reduced short-range immunoglobulin heavy chain (Igh) and T-cell receptor beta (Tcrb) D-to-J recombination

• Ubiquitin ligase activity:

  • The RING domain (aa 287-351) provides ubiquitin ligase activity that regulates RAG1 itself and potentially other targets

  • This activity enhances the efficiency and fidelity of the rearrangement reaction

• Protein stability regulation:

  • The non-core region controls RAG1 stability and expression levels

  • Deletion of this region can lead to elevated RAG1 expression compared to wild-type, resulting in increased off-target activity

Studies using various RAG1 mutants (including P326G affecting ubiquitin ligase activity and K233R affecting autoubiquitination) have demonstrated that these specific residues within the non-core region influence recombination efficiency and accuracy . The non-core region of RAG1 thus serves as a critical regulator that preserves V(D)J recombination fidelity by preventing inappropriate recombination events .

What are the molecular mechanisms by which partial RAG1 deficiency leads to autoimmunity?

Partial RAG1 deficiency leads to autoimmunity through several interconnected molecular mechanisms:

• Restricted primary B cell receptor (BCR) repertoire:

  • Reduced RAG activity results in a limited primary BCR repertoire that is enriched for autoreactivity

  • This restricted repertoire fails to undergo proper negative selection, allowing self-reactive clones to escape central tolerance mechanisms

• Impaired receptor editing:

  • Partial RAG deficiency compromises receptor editing, a key process for eliminating self-reactive B cells in the bone marrow

  • This defect allows autoreactive B cells to enter peripheral circulation

• Inflammatory microenvironment:

  • Partial RAG deficiency creates an inflammatory milieu with elevated B cell-activating factor (BAFF)

  • This environment promotes B cell dysregulation with widespread activation and remarkable extrafollicular maturation

• T-bet+ B cell accumulation:

  • Patients with partial RAG deficiency show expansion of T-bet+ B cells

  • These cells undergo somatic hypermutation outside germinal centers, leading to persistence and expansion of self-reactive clones

• Persistent antigenic stimulation:

  • The restricted TCR repertoire leads to inadequate clearance of antigens, resulting in persistent stimulation that drives further autoimmunity

  • This chronic stimulation, combined with TLR activation, broadens the autoantibody profile over time

The combination of these factors creates what researchers describe as a "RAG-dependent 'domino effect'" that progressively impacts tolerance stringency and B cell fate in the periphery, explaining the late-onset autoimmune manifestations observed in patients with partial RAG deficiency .

How can RAG1-deficient pigs be utilized as models for human immune disorders and regenerative medicine?

RAG1-deficient pigs offer several advantages as models for human immune disorders and regenerative medicine applications:

• Xenotransplantation research:

  • The absence of functional T and B cells in RAG1-deficient pigs prevents rejection of human cells and tissues

  • These animals can accept xenografts without immunosuppression, providing platforms for testing human cell therapies

• Modeling human SCID conditions:

  • RAG1-deficient pigs recapitulate the T-B-SCID phenotype seen in humans with RAG1 mutations

  • Their larger size and longer lifespan compared to mice allow for longitudinal studies more relevant to human disease progression

• Stem cell research applications:

  • These pigs can serve as recipients for human hematopoietic stem cell transplantation

  • Studies show successful engraftment of human cells, enabling investigation of human immune development in a physiologically relevant large animal model

• Testing gene therapy approaches:

  • RAG1-deficient pigs provide an ideal system for evaluating gene therapy strategies before human trials

  • The efficacy of lentiviral vectors carrying RAG1 can be assessed in a model that closely resembles human physiology

• Humanized pig models:

  • Combining RAG1 deficiency with other modifications (e.g., FAH knockout or expression of human cytokines) creates sophisticated humanized models

  • RAG2-/-IL2Rγ-/Y FAH-/- (RGFKO) pigs, for example, support human hepatocyte engraftment and expansion

Experimental data demonstrates the utility of these models, with successful generation of various RAG1-deficient pig lines showing consistent immunological phenotypes across studies . The physiological similarities between pigs and humans make these models particularly valuable for translational research aimed at developing therapies for human immunodeficiencies .

What are the key differences between RAG1 and RAG2 deficiencies in porcine models?

While both RAG1 and RAG2 deficiencies result in SCID phenotypes in pigs, several key differences have been observed:

• Developmental defects:

  • RAG1-deficient pigs show more severe thymic hypoplasia compared to RAG2-deficient counterparts in some studies

  • Both models demonstrate absence of mature B and T cells, but RAG1 knockouts may exhibit more profound lymphoid organ developmental defects

• Residual recombination activity:

  • Some RAG2-deficient models show trace V(D)J recombination activity not observed in complete RAG1 knockouts

  • This suggests RAG1 may retain minimal functionality even in the absence of RAG2 in certain contexts

• Phenotypic stability:

  • RAG1-deficient pigs tend to show more consistent and stable SCID phenotypes across generations

  • RAG2 deficiency phenotypes can show slightly more variability, particularly in terms of NK cell development

• Survival rates:

  • Studies report comparable survival rates for both RAG1 and RAG2 knockout piglets under controlled conditions

  • When maintained in specific pathogen-free environments, both models can survive beyond weaning with appropriate care

Table 1: Comparative analysis of RAG1 vs. RAG2 knockout pig models

*Gene editing efficiency refers to the percentage of correctly targeted cell colonies obtained after CRISPR/Cas9 editing.

What methodological challenges exist in designing catalytic RAG1 mutants for research applications?

Designing catalytic RAG1 mutants for research applications presents several methodological challenges that researchers must address:

• Identifying critical residues:

  • The core catalytic domain contains multiple conserved residues (D602, D710, E964) whose mutation can affect activity to varying degrees

  • Different mutations in the same residue can produce distinct phenotypes, requiring careful design

• Dominant-negative effects:

  • Some catalytic RAG1 mutants exhibit dominant-negative effects, inhibiting wild-type RAG1 activity

  • This property can be exploited for research but must be controlled to avoid unintended consequences

• Protein stability considerations:

  • Many RAG1 mutants show reduced stability compared to wild-type protein

  • Stability can be enhanced by replacing the N-terminus with fluorescent tags (e.g., EGFP) which increases protein half-life

• Expression level variability:

  • The expression level of RAG1 mutants is critical for phenotype manifestation

  • Transgenic animals often show variable expression across tissues and between individuals, complicating interpretation

• Conditional expression systems:

  • Developing conditional RAG1 mutant expression systems is challenging due to the complex regulation of RAG1 during lymphocyte development

  • Temporal control of expression requires sophisticated inducible systems that maintain physiological relevance

To address these challenges, researchers have developed approaches such as fusion with stabilizing domains, codon optimization, and careful titration of expression levels. For example, one study demonstrated that a triple mutant RAG1 (D602A/D710A/E964A) fused with EGFP showed a twofold increase in dominant-negative inhibitory capacity compared to non-stabilized mutants .

How do current gene correction approaches for RAG1 compare in terms of efficiency and safety profiles?

Current gene correction approaches for RAG1 show varying efficiency and safety profiles, with important considerations for researchers:

Research indicates that gene correction approaches that preserve the physiological regulation of RAG1 expression are preferable for clinical translation, as constitutive expression of RAG1 outside its normal developmental window could have deleterious effects on genomic stability .

What emerging technologies might improve the generation and characterization of partial RAG1 deficiency models?

Several emerging technologies show promise for advancing partial RAG1 deficiency models:

• Base editing and prime editing:

  • These technologies allow precise nucleotide changes without double-strand breaks

  • Particularly valuable for generating hypomorphic RAG1 mutations that mimic human variants

  • Can achieve higher efficiency than traditional HDR for introducing point mutations

• Single-cell multiomics:

  • Integrating single-cell transcriptomics, epigenomics, and repertoire analysis

  • Enables detailed characterization of altered lymphocyte development in partial RAG1 deficiency

  • Can identify cell-specific consequences of reduced RAG1 activity on immune repertoire formation

• Inducible RAG1 expression systems:

  • CRISPR interference (CRISPRi) or CRISPR activation (CRISPRa) to modulate endogenous RAG1 expression

  • Allows temporal control of RAG1 activity to study developmental stage-specific functions

  • Can create models with variable degrees of deficiency to better replicate human disease spectrum

• Humanized porcine models:

  • Introduction of human RAG1 variants into RAG1-deficient pigs

  • Creation of pigs expressing human cytokines to support development of human immune cells

  • These models would better recapitulate human disease and facilitate testing of human-specific therapies

• Organoid technologies:

  • Thymic and bone marrow organoids derived from RAG1-mutant cells

  • Allow high-throughput testing of correction strategies in controlled microenvironments

  • Artificial thymic organoids (ATOs) have already been used to evaluate RAG1 correction approaches

These technologies collectively promise to create more precise and physiologically relevant models of partial RAG1 deficiency, facilitating better understanding of disease mechanisms and development of targeted therapies.

How might understanding RAG1 regulatory mechanisms inform gene therapy approaches for immunodeficiencies?

Understanding RAG1 regulatory mechanisms has profound implications for gene therapy approaches for immunodeficiencies:

• Optimal promoter selection:

  • Knowledge of RAG1's complex developmental regulation informs the choice of promoters for gene therapy vectors

  • Endogenous or physiologically similar promoters prevent inappropriate expression and potential genomic instability

• Non-core region inclusion:

  • The regulatory functions of RAG1's non-core region suggest gene therapy vectors should include these domains

  • Complete RAG1 constructs with non-core regions show improved fidelity of V(D)J recombination compared to core-only constructs

• Targeting appropriate cell populations:

  • Understanding the developmental timing of RAG1 expression guides selection of optimal cell populations for therapy

  • Studies indicate earlier progenitors may be more effective targets than committed lymphoid cells

• Refining gene editing strategies:

  • Insights into RAG1 autoregulation inform the design of HDR templates

  • Preserving regulatory elements in knockin constructs maintains normal expression patterns

  • In-frame exonic insertions have shown superior results to intronic integration strategies

• Dose optimization:

  • Knowledge of RAG1 expression thresholds helps determine minimal effective doses

  • Studies indicate correction efficiency exceeding 1-10% of HSPCs may be sufficient to rescue immunodeficiency, reducing safety concerns associated with high vector doses

Current research demonstrates that gene therapy approaches that preserve physiological RAG1 regulation show the most promise for clinical translation, as they minimize risks associated with dysregulated expression while effectively rescuing immune function .

What potential applications exist for engineered RAG1 variants in synthetic biology and genome editing?

Engineered RAG1 variants offer intriguing applications in synthetic biology and genome editing:

• Programmable genome editing tools:

  • Engineered RAG1/RAG2 complexes with modified RSS recognition domains could serve as alternative site-specific nucleases

  • These could complement CRISPR-based systems for applications requiring different PAM requirements or cleavage patterns

• Controlled VDJ recombination:

  • Engineered RAG1 variants with altered specificity could direct recombination to predefined loci

  • Applications include generation of animals with predefined antibody repertoires for research or biotechnology

• Synthetic immune circuit design:

  • Modified RAG1 systems could be incorporated into synthetic genetic circuits in mammalian cells

  • These circuits could create programmable cellular responses based on DNA rearrangement instead of transcriptional control

• Biocontainment strategies:

  • RAG1-mediated genetic rearrangements could be engineered as biocontainment mechanisms

  • Programmed rearrangements could disable essential genes under specific conditions, providing biological containment for engineered organisms

• Directed evolution platforms:

  • Controlled RAG1-mediated recombination could generate diversity in protein-coding sequences

  • This approach could complement existing directed evolution methods by introducing diversity at specific predetermined sites

Early research already demonstrates the feasibility of some of these applications. For example, studies have shown that fusion of the RAG1 catalytic domain to programmable DNA-binding proteins can direct DNA cleavage to specific sites, suggesting the potential for developing RAG1-based genome editing tools with unique properties compared to existing nucleases .